Body fluids test - lectures 7-11

Other cells seen in CSF

-Blasts (ALL): chemotherapeutic drugs are blocked from reaching the brain by the blood-brain barrier - Macrophages & monocytes -Erythrophages & siderophages (brain bleed) -Hemosiderin-laden macrophages (old hemorrhage) -Choroid plexus cells (cells lining the CNS) -Bone cartilage cells -Ependymal cells (ventricular lining cells) -Eosinophils (↑malfunctioning ventricular shunt)

Processing and storing body fluids

-Body fluids should be analyzed as soon as possible after collection, especially CSF (STAT sample) -If a cell count is ordered on a fluid other than CSF, it is collected in a tube w/ anticoagulants (EDTA or Heparin) -Most fluid require cytospin prep for accurate cell differentiation -Some cell counts are performed using a hemocytometer, some use automated analyzers

Bacterial Meningitis

-Characterized by a high WBC and increase in neutrophils -Leads to more permeable blood-brain barrier due to increased inflammation -Hallmark finding = reduced glucose levels in CSF Possible causes: -Neisseria meningitidis (rash on extremities is a medical emergency) -Strep pneumo -Mycobacterium tubercolosis (spider web clot may be seen in tube)

Lab tests on serous fluid

-Chemical analysis -Culture & sensitivity -Immunologic assays -Molecular assays -Cytologic testing -Hematologic exam (macroscopic/microscopic)

Seminal fluid speciment collection

-Collected following a period of sexual abstinence (<7 & >2 days) -Collect 2-3 samples no less than 7 days apart - no more than 3 weeks apart -Collect at home in special container. Time is noted on container -Specimen is maintained at room temp and delivered to lab w/in 1 hour of collection -Specimens are collected by masturbation or by use of special condoms

Rationale for seminal fluid analysis

-Fertility evaluation -Post-vasectomy evaluation

Cytospin time and speed

-For most fluids, pipet 0.25mL (or 5 drops undiluted fluid) into funnel -Spin 1000rpm for 10 minutes -If nucleated cell count on the undiluted fluid is >200/μl, divide the cell count by 100 to get a dilution factor -If a dilution is needed, prepare with saline. Transfer 5 drops of diluted fluid into funnel

Monosodium urate crystals (MSU)

-Gout -Negatively birefringent -Needle-like, intracellular crystals -Yellow in color when parallel to the slow axis of the compensator -Blue in color when perpendicular to the slow axis of the compensator

Challenges for labs testing body fluids

-Healthy subjects do not normally donate body fluids due to the invasive nature of the collection process -This contributes to a lack of matrix-matched quality control materials, information on stability and storage, etc. -Published data that is available may not be transferable to the lab's testing protocol and patient population -Some labs send body fluids out for testing

Conditions diagnosed with CSF

-Meningitis -Encephalitis -Syphilis -Brain abscess -Multiple sclerosis -Subarachnoid hemorrhage -Guillain-Barre -Acute leukemia and tumors of spinal cord & brain

Seminal fluid color and appearance

-Normal = gray-white, translucent -Increased turbidity may indicate WBC -Red coloration may indicate RBC -Yellow coloration observed if specimen is contaminated with urine (urine is toxic to sperm and may affect the result of motility test)

Synovial fluid crystal analysis procedure

-Place one drop of synovial fluid in EDTA on a glass slide & coverslip -Position the slide on the microscope -Focus at 10x, examine at 40x -Look for crystals, if present slide the red compensator into position and observe for birefringence

3 types of serous fluid

-Pleural fluid -Pericardial fluid -Peritoneal fluid fluids collected by physician by needle aspiration is sterile tubes with EDTA

Primary causes of effusion

-Poor perfusion (congestive heart failure (CHF)) -Decreased osmotic pressure (hypoproteinemia) -Increased capillary fragility (infection/inflammation) -Lymphatic obstruction (tumors)

Calcium pyprophosphate dehydrate crystals (CPPD)

-Pseudo-gout -(positively) birefringent -Rhombic shaped crystals -Blue in color when parallel to the slow axis of the compensator -Yellow in color when perpendicular to the slow axis of the compensator

Seminal fluid

-Semen -Fluid in which sperm are distributed (transport medium) -Produced in seminal vesicles -Provides nutrients for preservation of sperm

Peritoneal fluid

-fluid surrounding the abdomen. -aka ascetic fluid Removed by Paracentesis

CSF cell count

0-5 WBCs/μL, of these no more than one should be a PMN cell >5 WBCs/μL - suspected meningitis <100 cells on cytospin prep, count cells and report as whole number >100 cells on cytospin prep, 100 cells should be differentiated and reported as a percentage.

Sperm morphology categories

2 categories: normal & abnormal Abnormal forms are described with descriptive comments Normal = 50% or more normal forms Less than 50% normal forms may be associated with infertility Some labs use Kruger scoring system

Parts of the polarizing microscope

3 basic parts -Polarizer (under stage) -Analyzer (under oculars) -Compensator (under analyzer to the side)

Cytocentrifugation

A centrifuge used to concentrate cells in body fluid (20x concentration) Deposits a monolayer of cells into a circular area on a slide, air dry, stain with Wright's stain, read microscopically at 100x (oif) to differentiate Prepared slide is referred to as a cytospin preparation

Sperm fructose

A low sperm count can be associated with a lack of support medium produced by the seminal vesicles. Reflected in low to absent fructose level. Low fructose levels are also seen in obstruction of the ejaculatory duct Ref range = 150-600mg/dL

Birefringence

A physical characteristic of some crystals Can be used to distinguish the crystals associated with Gout and Pseudogout CPPD crystals: positive, pseudogout MSU crystals: negative, gout

Malignant cells

A primary concern in evaluating all serous effusions Differentiation is difficult (as with mesothelial cells) When is doubt - ASK!! Any suspicious cells may be reviewed by the hematology supervisor, cytology dept., or a pathologist

Tau isoform of transferrin

A protein found in CSF that is not found in blood, other body fluids, or secretions Elevated levels support the diagnosis of a skull fracture

Cell count in synovial fluid

A small amount of hyaluronidase should be added to synovial fluid just prior to performing the cell count or cytospin concentration. This liquifies the fluid Normal cells in synovial fluid include lymphocytes, macrophages, & synovial cells Synovial cells resemble mesothelial cells in appearance

Effusions

Abnormal accumulation of serous fluid Result from the disruption in the balance of pressure due to infection or inflammation

Clot formation in CSF

Abnormal finding Occurs due to the presence of increased fibrinogen typically introduced as the result of a traumatic tap

CSF total normal volume

Adults: 90-150mL Neonates: 10-60mL

Gout

Arthritis: synovial fluid is poorer solvent for sodium urate than plasma, so with hyperuricemia Urates in joint fluid crystalize, particularly in ankle due to lower temperature. Crystals develop in synovial lining cells, stimulates formation of antibodies which accelerates formation of new crystals Release of crystals attracts neutrophils and complement (generates c3a, c5a, attracts more neutrophils) Releases free radical and lysosomal enzymes

Synovial fluid analysis

Assists in the diagnosis of arthritis by type and includes White cell count Crystal analysis Glucose level Culture/Gram stain

Chylous

Body fluid that is white in color (containing lipids)

Off label body fluids

Body fluids that are not derived from blood or urine. -CSF -Serous fluids (pleural, percardial, pertoneal) -Synovial fluid -Fluids obtained by drainage or washing (BAL)

Locations fluids normally present

CSF, Synovial, Pericardial In other location, the accumulation of fluid is a pathologic finding In other cases, fluids obtained by the collection of washings

Zollinger-Ellison Syndrome (ZES)

Characterized by the development of a tumor (gastrinoma) or tumors that secrete excessive levels of gastrin

Sweat

Chemical analysis performed to investigate chloride levels for suspected cases of cystic fibrosis Sweat glands affected by cystic fibrosis causing excessive chloride to be produced

Normal appearance of CSF

Clear & colorless (resembles water) Pink or red indicates traumatic tap or subarachnoid hemorrhage

Synovial fluid collection

Collected by a physician by inserting a needle into the space surrounding a moveable joint Collected in EDTA (lavender top) when crystal analysis is requested

CSF collection tubes

Collected in a minimum of 3 sequentially numbered capped tubes containing at least 1mL each. No anticoagulant, always performed STAT. Tube 1: Microbiology for Gram stain , C & S Tube 2: Chemistry (protein, glucose Tube 3: Hematology (cell count & diff) If a 4th tube is collected, it's usually sent to serology for VDRL. If a 4th tube is sent but no test are ordered for it, freeze it.

BAL physical examination

Color may be colorless, milky white, light brown-beige, or red Clarity may be clear, hazy, cloudy, or turbid Volume is measured and reported

Hemacytometer sperm count

Count all sperm in blocks 1, 2, 3, 4, 5 Compute the TOTAL number. Do not average or use the standard formula for computing cell counts Add 6 zeros to the number and write "/mL" after the number

Sperm count

Counted with the aid of a hemacytometer in the RBC counting area A 1:20 dilution is prepared using sodium carbonate as the diluent Reported as #/mL Ref range = 20-250 million/mL A sperm count <20,000,000 is associated with infertility

3 meningeal membranes

Covers the brain and spinal cord. -Pia mater -Arachnoid mater -Dura mater

Hematology tests on serous fluid

Differential count is the most useful Most common cell types seen in serous fluids: -Macrophages (64-80%) -Neutrophils (1-2%) -Lymphocytes (18-30%) -Mesothelial cells -Plasma cells -Malignant cells

Pseudogout

Distributed equally among males and females Seen most often in persons >50 yrs of age Not joint specific - may affect any joint Characterized by the finding of crystals that are positively birefringent (CPPD)

Exudate

Effusion that results from a condition that directly impacts the serous membrane Examples: -Infection -Inflammation -Malignancy sp. grav = >1.016

Transudates

Effusion that results from a systemic disorder that disrupts the regulation of fluid filtration and re-absorption Common causes: -Poor perfusion (Congestive heart failure CHF) -Hypoproteinemia (nephrotic syndrome) -Liver/Kidney disease -Accompanied by general edema Treatment aimed at addressing the underlying cause sp. grav = <1.016

Oligoclonal

Electrophoreses bands present in CSF but absent in serum

Sperm morphology

Evaluated by placing about 10μL of seminal fluid on a glass slide and smearing out slightly, air dry, and stain with Wright's stain Examine at least 100 sperm microscopically at 100x (oil) looking at -Head -Neckpiece -Mid-piece -Tail

Sperm motility: grade 4

Fast & forward progression where sperm move in a straight direction

steatorrhea

Fat or fatty acids in stool Too much fat in stool is a sign of malabsorption

Seminal fluid viscosity

Fresh semen is too viscous to be analyzed. Allow specimen to liquefy for at least 30 minutes following collection Incompletely liquefied specimens are clumped and thick After 30min, reported as slightly viscous, viscous, or highly viscous Normal = viscous high viscosity impedes movement

Cystic fibrosis

Genetic disorder that affects the mucus glands, especially the lungs and pancreas, causing production of a thick, sticky mucus that clogs the lungs and blocks the pancreas Caused by genetic mutation called delta-F508

Bile

Imparts a green color to body fluid Brown to dark green fluid produced by the liver, stored in the gallbladder, and released into the intestines when we eat Gall is a synonym for bile

Bacteria in CSF

In bacterial meningitis, the predominant WBC seen is the neutrophil. Look for intracellular bacteria and report if seen

Blood-brain barrier

In normal adults, is well developed and restricts the flow of macromolecules such as blood cells and proteins. A breach is evidenced by CSF/serum albumin levels >9

4 general conditions diagnosed by body fluids test

Infection Hemorrhage Malignancy Inflammation

Meningitis

Infection or inflammation of the meninges, especially the pia mater and the arachnoid Often bacterial in nature but may also be caused by viruses and fungi

Red compensator

Inserted between the polarizer and the analyzer Turns the background of the viewing field red/purple Crystals in the sample will appear blue or yellow Crystal orientation can be noted in relation to the compensator marked with an arrow. The long axis of the crystal is aligned with the orienting arrow

BAL cell differential

Involves cytocentrifugation Macrophages are most common cells seen, 56-80% or all nucleated cells Lymphocytes comprise 1-15%. >25% lymphs = suspected granulomatous lung disease >50% = suspected hypersensitivity pneumonia or non-specific interstitial pneumonia Neutrophils comprise 3% or less. Increase = infection Eosinophils comprise 1-2%. Increased in eosinophilic pneumonia & parasitic infection

BAL specimen handling

Keep specimen at room temp during transport If delivery to lab is expected to exceed 30mins, store on ice Cell counts should be performed within 1 hour of collection Specimens are unacceptable for testing after 24 hours

Bronchoaveolar Lavage (BAL)

Lani Collins described BAL as "fluid that isn't a fluid" "fluid" is obtained by introducing warm saline into the lungs through a bronchoscope in 20mL aliquots follows by the aspiration of the saline Total of 100-120mL of saline infused with a recovery of 40-60mL The "fluid" is sent to the lab for microbiological & cytological testing Occasionally, a cell count diff is requested

Normal cells seen in CSF

Lymphocytes (predominant in adults) Mononuclear cells (predominant in neonates)

Seminal fluid analysis

Macroscopic -Color & appearance -Volume -Viscosity -pH Microscopic -Sperm count -Sperm motility -Sperm morphology

Mesothelias cells vs Malignant cells

Mesothelial: -Low N:C ratio -Smooth -Windows between cells -Small nucleoli if present -Normal to reactive -Cytoplasm is dark blue Malignant: -High N:C ratio -Irregular/jagged -Obscured window -Prominent/irregular nucleoli -Immature/atypical -Basophilic, may be vacuolated

viral meningitis

More common in children May cause fever, lethargy, poor feeding Predominant WBC seen is the lymphocyte Some lymphocytes seen may be small or large, others may have plasmacytoid appearance

Gout characterisitc

More common in males but may be seen in children and is not common in post-menopausal females Seen more in those who eat meat & drink beer Associated with hyperuricemia Not joint specific but often affects the big toe first Characterized by the finding of crystals that are negatively birefringent (MSU)

Abnormal sperm

Normal Condensed acrosome Small head Large head Double head Double tail Abnormal middle-piece

Seminal fluid volume

Normal = 2-5mL Measured by pouring the specimen into a graduated conical centrifuge tube (like the type used in urinalysis) May also be measured by drawing the full volume of sample in the cup up in a disposable 10mL serological pipet. Requires excellent manual pipetting technique

Sperm motility

Normal sperm exhibit forward progressive movement (critical for fertility) Assessed by examining a well-mixed liquefied specimen within 1 hour of collection A drop of seminal fluid (10μL) is placed on a slide, coverslipped with 22x22mm coverslip, settle for 1min, examined microscopically at 40x

Sperm anatomy

Normal sperm have oval-shaped head, neck piece, mid-piece, and tail Normal: -Smooth, oval-shaped head, 5-6 micrometers long, 2.5-3.5 micrometers wide (less than the size of a needle point) -Well-defined cap (acrosome) that covers 40-70% of the head -No visible abnormality of neck, mid-piece, or tail -No fluid droplets in the sperm head that are bigger than one half of the sperm head size

Serous fluid cell count & differential

Normally performed on fluid collected in EDTA RBC & WBC counts are not routinely performed on serous fluids. When ordered, run on an electronic cell counter WBC > 1000/μL, RBC > 100,000/μL indicates exudate Differential cell counts are performed on cytospin preparations stained with Wright's stain

Cell count and differential

Performed on undiluted CSF with a hemocytometer RBC's and WBC's are counted in 10 sq.mm (9 on one side of the hemocytometer, 1 on the other) The process is repeated and the results are averaged

Conditions diagnosed with BAL

Pneumocystis jirovecii (formally carinii) Diffuse lung disease (sarcoidosis) Exclude other diseases in patients with pulmonary fibrosis

Crystal analysis with polarized light

Polarized light used to examine synovial fluid for crystals (polarizing microscope) Polarizing microscopes utilize red compensators integrated into the housing of the polarizing filter Incident light encounters the polarizing filter and exits as polarized light When the light strikes the unknown crystal, the color of the light and its orientation assist in crystal identification

Thoracentesis

Procedure to remove fluid from the space between the lungs and the chest wall (pleural fluid)

Spermatogenesis

Process of sperm production

BAL cell count

RBC & WBC counts are performed on a hemacytometer BMP LeukoChek system may be used if WBC dilution is necessary RBC dilutions may be prepared with isotonic saline Automated cell counters may not be used ↑ neutrophils = infection ↑ eosinophils = allergic reaction, parasitic infection or eosinophilic pneumonia ↑lymphs = viral pneumonia, connective tissue disorder, or lymphoma

Transudate vs Exudates chart

Remember from chart Transudate: -Appearance = clear -All other characteristics are "less than" (<) except for Serum ascites albumin gradient (>) -No spontaneous clotting Exudate: -Appearance = Cloudy -All other characteristics are "greater than" (>) except for Serum ascites albumin gradient (<) -Possible spontaneous clotting

Serous fluid physical appearance

Resembles serum in appearance (clear/pale yellow)

Sperm production site

Semi-ferous tubules of the testis

Arthritis Categories

Septic arthritis (infection) Inflammatory arthritis: -Rheumatoid arthritis -Gout (MSU) -Pseudogout (CPPD) -Lyme disease Lupus (systemic, autoimmune disorder) Osteoarthritis (degenerative arthritis)

Transudates & exudates

Serous fluid classification Distinction is made by lab tests Correctly classifying effusions can help determine the underlying cause and allow physician to treat the patient appropriately Transudates: results from disease process outside the body Exudates: results from disease process inside the body Transudate sp. grav = <1.016 Exudate sp. grav = >1.016

pH of seminal fluid

Should be checked within one hour of specimen collection Ordinary pH paper used to determine Normal semen is slightly alkaline = 7.2-8.0 Increased observed in infection Decreased observed in increased prostatic fluid output, urine contamination, with obstruction of vas deferens, or with poorly developed seminal vesicles

Cholesterol crystals in synovial fluid

Signifies inflammation Large extracellular plates with notched edges

Sperm motility: grade 1

Sperm move but fail to progress forward

Sperm motility: grade 3

Sperm move forward but at a slower speed and/or in a curved direction

Sperm motility: grade 2

Sperm move slowly and in a poorly defined direction

Sperm motility: grade 0

Sperm show no signs of movement

Xanthochromic

Supernatant fluid that is pink, orange, or yellow color to CSF even after centrifugation Results from the breakdown of hemoglobin. Associated with intracranial hemorrhage. Present in the CSF of more than 90% of patients within 12 hours of onset subarachnoid hemorrhage

Serous cavities

Surround various organs Serous fluids fill the space between body wall and organ Function as a lubricant to prevent friction between the body wall and the organ Lined with mesothelial cells

Synovial fluid

Surrounds a moveable joint Used for crystal analysis in differentiating gout and pseudogout Place 1 drop on a glass slide, coverslip, examine on high power using polarized light Collected to determine the cause of painful swelling of a joint, fluid accumulation around a joint, joint stiffness, or joint inflammation (arthritis)

Pilocarpine iontophoresis

Sweat test for cystic fibrosis Patient induced to sweat by placing pilocarpine nitrite on the skin. Sweat is collected on to filter paper, eluted & analyzed for it's chloride concentration as part of the sweat conductivity test Positive result = sweat chloride >60 mEq/L (60 mmol/L

Type II pneumocytes

The cell responsible for the production and secretion of surfactant (the molecule that reduces the surface tension of pulmonary fluids and contributes to the elastic properties of the lungs) Pneumocystis pneumonia (PCP) is a serious infection caused by the fungus Pneumocystis jirovecii. Most people who get PCP have a medical condition that weakens their immune system, like HIV/AIDS, or take medicines (such as corticosteroids) that lower the body's ability to fight germs and sickness.

Relative position and use of polarizing microscope parts

The compensator (comp) is positioned between the analyzer (ana) and the polarizer (pol) Crystals seen in the synovial fluid are observed relative to the position of the compensator The compensator runs in a southeast to northwest direction ↖

Multiple sclerosis

The finding of 2 or more CSF specific oligoclonal bands in the gamma region of the CSF electrophoresis is consistent with this disease

Cerebrospinal fluid (CSF)

The primary purpose of CSF is to cushion the brain and spinal cord. CSF is found in the subarachnoid space between the arachnoid and dura mater Sample obtained by lumbar puncture

malabsorption

Too much fat in stool is a sign that food is moving through intestine without being broken down and absorbed properly Fecal fat test to determine

traumatic tap vs subarachnoid hemorrhage

Traumatic: -Progressively less blood in the samples from 1st tube to subsequent tubes -Supernatant fluid is clear after centrifugation Subarachnoid hemorrhage: -Blood is evenly distributed -Supernatant is xanthochromic

BAL special precautions

Usually performed on a hospitalized patient who has non-resolving pneumonia in an attempt to identify organisms that are not responding to conventional antibiotic therapy Also done to detect malignancy Organisms in these fluids are frequently airborne A mask should be worn when performing the cell count and diff and performed under the hood.

Sperm motility grading criteria

WHO criteria Keys on diff count labeled 0-4, count 100 sperm Scores assigned to evaluate progression (forward movement) Normal: report % with each score. 50% or more are scored 2 or higher

Post-vasectomy semen analysis

Wet-prep is prepared and examined microscopically using bright-field or phase contrast microscopy. Checked for viable sperm, if none seen, the specimen is centrifuged for 10 minutes and the sediment examined Report results as "sperm seen" or "no sperm seen" No sperm should be seen

Serous fluid

an ultrafiltrate of plasma, maintained by pressure forces - a tightly controlled process that allows for the continuous exchange of fluid from the cavity to surrounding capillaries.

Pericardial fluid

fluid surrounding the heart

Pleural fluid

fluid surrounding the lungs aka thoracentesis fluid Removed by thoracentesis

BAL cell count: other significant findings

↑ macrophages with tar: ↑ in smokers ↑ erythrophages if there has been a hemorrhage in lungs in preceding 12 hours Ciliated epithelial cells (columnar epithelial cells) Type II pneumocytes ↑ in ARDS Pneumocytis