Pages From Qiuck Card 1

SECTION 1

Clinical Laboratory Practice Review

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Credentialing

Process Definition Exumples

Accreditation A voluntary process of external peer review American Association of Blood Banks in which a private, nongovernmental agency College of American Pathologists

i grants puhlic recognition to an institution Joint Cornmission on Accreditation of Health that meets certain standards Care Organizations

National Accrediting Agency for Clinical Laboratory Sciences

Certification A voluntary process by which a nongovern- American Medical Technologists mental agency grants recognition to an American Society of Clinical Pathologists individual who has met certain educational International Society for Clinical Laboratory requirements and demonstrated entry level Technology competency by examination National Credentialing Agency for Laboratory

Personnel

Licensure A mandatory process by which a state grants Licensure of laboratory personnel is required in permission to an individual or organization California, Florida, Georgia, Hawaii, Louisiana, to engage in a given occupation or business Montana, Nevada, North Dakota, Rhode Island,

Tennessee, and West Virginia. Many states require licensure of clinical laboratories.

Kegistration A process by which qualified individuals are listed on an official roster maintained by a governmental or nongovernmental agency

2 CLINICAL LABORATORY PRACTICE REVIEW

Regulatory Agencies

Agency Authority

Department of Transportation Federal agency that regulates packaging, labeling, and transportation of (DOT) biological products

Envirorlmental Protection Federal agency that regulates disposal of toxic chemical and biohazardous Agency (EPA) wastes

- - - - ~ - - - - - - - - - - - ~~~ ~ - ~ ~

Food and Drug Administration Federal agency that regulates market entry of instruments and reagents, (FDA) regulates production of donor blood and components, and licenses blood banks

Health Care Financing Federal agency that administers Medicare and federal portion of Medicaid Administration program. Writes regulations for and enforces Clinical Laboratory Amendments (HCFA) of 1988 (CLIA '88).

Department of Health and Federal agency that interprets and implements federal regulations related to Human Services (HHS) healthcare. Includes HCFA, FDA, and CDC.

Department of Public Health State agency that develops rules and regulations to implement a state's public health laws

Nuclear Regulatory Commission Federal agency that licenses laboratories that use radionucleotides (NRC)

Occupational Safety and Federal agency that regulates employee safety in the workplace Health Administration (OSHA)

Substance Abuse and Mental Federal agency that issues mandatory guidelines for laboratories performing Health Services Administration forensic toxicology. Formerly National Institute on Drug Abuse (NIDA). (SAMHSA)

4 CLINICAL LABORATORY PRACTICE REVIEW 3

C - - - -- - -

Agencies That Issue Guidelines/Standards

Agency Guidelines/Stnndards

Amer-ican Association of Blood Batiks (AABK) Technical standards for blood banks

Centers for Disease Control and Prevention (CDC) Standards and guidelines for I~ospitals and laboratories, primarily related to infei.tion control and safe work practices

-- International Organization for Standardization (ISO) Standards to facilitate the international exchange oi goods

and srrvices. Developed through a volurltary worldwide consensus process (e.g., IS0 9000 provides a framrwork for quality management and quality assurarlce).

Sational Committee for Clinical Lal~oratory S t a ~ ~ d a r d s on all aspects of laboratory practlce developed Sta~ldards (KCCLS) through a volulltary consensus pi-ocess

4 CLINICAL 1,ABOKATORY PRACTICE REVIEW

Clinical Laboratory Improvement Amendments of 1988 (CLIA '88)

All laboratories that examine human specimens for diagnosis, prevention, or treatment Applies to of disease

Exernptioils Work done for lcgal or cmployment eligibility purposes; research not used for patient care; VA, armed forces, Substance Abuse and Mental Health Services Admirlistration

Levels of co~l~plexi ty Waived, provider-performed microscopy, moderate complexity, high complexity

Administered by Health Care Finance Administration (HCFA)

Areas for compliance Personnel qualifications/competency assessment, patient test management, quality control, quality assurance, proficiency tesling

CLINICAL LABORATORY PRACTICE REVIEW 5 1

OSHA Standards

Yeur Standard Explanation I I

1984 Respiratory Protection Stdndard Requires a respiratory protection plan I !

1987 Hazdrd Communication Standard Requires elnplovers to inform employees about hazardous I

"Right-to-Know Law" substances ill the workplace and to educate and train them I in safe and proper liandling I

1 1991 Occupational Exposures to Requires a chemical hygiene plan to minimize pel.soilnel

Hazardous Chemicals in exposures to hazardous chemicals Laboratories-"Ldboratory Standard"

1991 Bloodburne Pathogens Standard hlandates strirlgent work practices and procedures to I minimize worker exposure to bloodborile pathogens 1

I 1992 (revised) For~naldehyde Standard Requires monitoring of forinaldehyde exposure, engineering I

controls, persolla1 protective equipment, training, emergency 1 action plan

1


Bloodborne Pathogens I

Specimens That Are Usi~ally Not Infectious I

Specimens Tlrat Are Potentially Infectious [Unless Visibly Bloody) I I

I

Blood Feces I

Pus and purulent fluids Nasal secretions I I

Semen Sputurn I

Vagirlal secretions Sweat I

Cerebrospinal fluid Tears Pleural fluid Urine Peritorleal fluid Vonlitus Pericardial fluid

1 A~nniotic fluid Breast milk I

I

CLINICAL LABORATORY PRACTICE REVIEW 7

Hazard Categories of Chemicals

Classification Example Effect Comments

Corrosives Glacial acetic acid, Visible destructio~l of human Chemicdis with pH <2 or >12. Separate hydrochloric acid, tissue upon contact. Can produce inorganic acids from organic acids. sodium hydroxide injury upon inhalation or contact. Concentrated acids and bases can

generate large amounts of beat when mixed with water.

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Toxic substances Cy'mides, sulfides I~lteriere with metabolic pro- Threshold limit values [TtWs) indicate cesses when ingested, inhaled, the amount an individual can be 01- absorbed through the skin exposed to without adverse effect.

Carcinogens Renzidine, Capable of causing cancer OSHA requires ir~onitoring of exposuri. fornialdehyde to formaldehyde.

Mutagens a n d Benzene, lead, Mutagells induce genetic Special precautions during pregnancy teratogens mercury, ~.adio- mutatiolls. Teratogens cause

active material, physical defects in developing toluene embryo.

lg~litahles Acetone, alcohols, Fire Flamrnahles have a flashpoint below ether, xylene 100°F. Combustibles have a flashpoint at

or above I0O"F. The flasl~point 1s the lowest temperature that produces sufficient vapor lo form an ignitable mixture at the surface of the liquid. A hear source, such as an electrical spark or open flame, must be present to ignite tlie vapor.

Corltinued 8 CLINICAL LABORATORY PRACTICE REL'IEW

Hazard Categories of Chemicals Continued

Classification Example Effect Comments

Reactives Ether, perchloric Explosion Ethyl and isopropyl ether form explosive peroxides on acid, picric acid, exposure to air or light; must be stored in an explosion- sodium azide proof refrigerator. Perchloric acid should be separated

from other acids; niay react explosively with organic compounds, including acetic acid. Picric acid becomes shock- sensitive when dehydrated and is a more powerful explosive than TNT. Pouring sodium azide solutions down the drain can cause explosive lead or copper azides to form from plumbing.


Hazard Identification System National Fire Protection Association (NFPA)

Hazard Svrnbol 0 I 2 3 4

Health Blue diamond No unusual Caution: May Warning: May hazard cause irritation be harmful if

inhaled or absorbed

Warning: Corrosive or toxic. Avoid skin contact or inhalation.

Danger: May be fatal on short exposure. Special- ized protective equipment required.

Flamn~ability Red diamond Not Combustible Caution: combustible if heated Combustible

liquid. Flash point of 100-200°F.

Warning: Flammable liquid. Flash point below 100°F.

Danger: Flammable gas or extremely flammable liquid

Reactivity/ Yellow Stable: Not Caution: May Warning: Danger: May Danger: stability dianiond reactive react if heated, Unstable, or be explosive Explosive

when mixed or mixed with may react if if spark occurs, material at with water water mixed with if heated room

water under confine- temperature ment, or mixed with water

Special White hazard diamond information


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Storage of Chemicals

Cherniml Category Examples Storage Guidelines

Acids Organic: formic, glacial acetic, citric Store below counter level or in acid cabinets. Inorganic: hydrochloric, nitric, sulfuric Separate from flammable and combustible material. Oxidizing: chromic, nitric, perchloric, Separate organic acids from inorganic acids.

sulfuric Separate oxidizing acids from organic acids. Separate from bases and active metals (e.g., sodium,

potassium, magnesium) .

Bases Ammonium hydroxide, potassium Separate from acids. hydroxide, sodium hydroxide Store inorganic hydroxides in polyethylene containers.

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Flammables Acetone, alcohols, xylene Store in approved safety cans or cabinets. No more than 60 gallons permitted in a single flammable

storage cabinet. Keep away from oxidizing acids and oxidizers.

pp - - - - - - - - - - - -

Oxidizers Nitric acid, perchloric acid, sulfuric Keep away from reducing agents (e.g., zinc, alkaline acid, acetic acid, potassium chloride, metals, formic acid). hydrogen peroxide Keep away from flammable and combustible materials.

- - -

Water- Sodium, potassium reactive chemicals

Keep away from water. Store in a dry, cool place.


Fire Safety

Class of Fire Combustible Material Extinguishers to Use Comments

A Cloth, wood, paper Pressurized water (A) Do not use pressurized water on electrical fires Dry chemical (ABC) or burning liquids.

B Flammable liquids Dry chemical (ABC) and gases Carbon dioxide (BC)

C Operating electrical Dry chemical (ABC) Dry chemical may cause electrical equipment equipment Carbon dioxide (BC) damage.

Halon Halon is first choice for computer and electrical equipment fires. Carbon dioxide is second choice.

D Combustible/ Leave to professional reactive metals firefighters.


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Commonly Used Anticoagulants

Stopper Anticoagulant Color Mode of Action Examples of Use Comments

EDTA (Na, or K,) Lavender Chelates Ca2+ CBCs, differentials, Prevents platelets from clumping. erythrocyte sedi- Causes minirnal morphologic changes mentation rate (ESR) in WBCs. ntbe should be at least half

full.

Heparin (Na, Li, Green Neutralizes Many chemistries, Best anticoagulant for prevention of or NH,) thrombin osmotic fragility, hemolysis. Not satisfactory for

plasma hemoglobin, differentials; causes a blue background. blood gases

Sodium citrate Light Blue Binds Ca2+ Most coagulation 3.2% or 3.8%. Preserves labile clotting tests factors. Tube must be filled completely

to achieve 9:l blood-to-anticoagulant ratio. Reduce anticoagulant for hematocrits over 55%.

Sodium fluoride/ Gray Oxalate binds Glucose, lactic acid Preserves glucose for 24 hours at room potassium oxalate Ca2+. Fluoride temperature and 48 hours at 4OC. Ox-

inhibits alate distorts cellular morphology; glycolysis. don't use for differentials.

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Lithium Gray Iodoacetate Glucose, lactic acid Used to preserve glucose and stabilize iodoacetate/ inhibits lactic acid levels. lithium heparin glycolysis.


Tests Requiring a Fasting Specimen Fasting blood sugar

Glucose tolerance test

Riglycerides

Lipid panel

Gastrin

Insulin


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Recommended Order of Drawing/Filling Tubes IB Procedure Order

Drawing evacuated tubes Sterile, red, light blue, green, lavender, gray

Filling evacuated tubes from syringe Sterile, light blue, lavender, green, gray, red

Filling microcontainers from finger-/heel-stick Lavender, green, red


- -- --- - ---

- - Situations in Phlebotomy

Situation Action

IV or blood transfusion running Never draw above an IV site. Use opposite arm or perform fingerstick, if possible. Otherwise, turn off IV for at least 2 minutes, apply tourniquet below IV site, select vein other than the one with the IV, draw and discard first 5 mL. Note that blood was taken from IV arm. Don't draw from an IV site for 1 hour after IV is discontinued.

Fistula Draw from opposite arm.

Indwelling lines and catheters, Usually not drawn by lab. The first 5 mL of blood drawn should be discarded. heparin locks, cannulas Lab may draw below heparin lock if nothing is being infused.

Sclerosed veins Select another site.

Hematoma Draw below.

Streptokinase/Tissue plasminogen Minimize venipunctures. Hold pressure until bleeding has stopped. activator (TPA)

Edema Select another site.

Scars, burns, tatoos Select another site. - ~

Mastectomy Draw from opposite arm.

Patient refuses Try to persuade. If unsuccessful, respect wishes and notify nurse. Never draw without consent. Charges of assault and battery could result.

Unidentified patient Ask nurse to properly identify patient before drawing.


-- - - Specimens Requiring Special Handling

Requirement Tests Comments

Chilling Arterial blood gases, adrenocorticotropic Place in crushed ice or a mixture of ice and hormone (ACTH), ammonia, gastrin, water. Do not use ice cubes alone because glucagon, lactic acid, parathyroid RBCs may lyse. hormone (PTH) , renin

Warming Cold agglutinins, cryoglobulins Use 37°C heat block, heel warmer, or hold in hand.

Protection from light Bilirubin, carotene, vitamin A, Wrap in aluminum foil. vitamin B,,

Chain of custody Any test used as evidence in legal Chain of custody form; lock boxes may be proceedings; e.g., blood alcohol, drug required. screens, DNA analysis

., CLINICAL LABORATORY PRACTICE REVIEW 17

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Specimen Processing Serum or plasma should be separated from cells within 2 hours of collection (unless collected in a gel separator tube).

Allow red top tubes to clot sufficiently (20-30 minutes) before centrifugation to avoid fibrin strands.

Centrifuge 1025 minutes at 1000-1200 X g.

Keep tubes capped during centrifugation to avoid loss of CO,, change of pH, evaporation, or aerosol formation.

Lipemic specimens can be ultracentrifuged at 105g to remove chylomicrons (triglycerides).

If analysis will be delayed more than 5 hours, serum or plasma for most tests can be capped and stored at 4°C. Some analytes should be frozen. (Do not freeze whole blood.) Avoid repeated freezing and thawing.

Specimens for lactate dehydrogenase (LD) should be kept at room temperature.

For acid phosphatase, add citrate (10 mg/mL) and freeze.

Glucose is stable in serum separator tubes for 24 hours and in sodium fluoride tubes for 24 hours at room temperature or 48 hours at 4°C.

Examples of Criteria for Specimen Rejection Missing or inadequate label

Collected at wrong time

Collected in wrong tube

Insufficient specimen

Inadequate volume of blood in anticoagulant tube

Exposure to temperature extremes

Prolonged transit

Clots in CBC tube

Hemolysis (depending on test ordered)

Lipemia (depending on test ordered)

CLINICAL LABORATORY PRACTICE REVIEW 19 I L

Centrifuge Fast Facts

Relative centrifugal force Force acting on sample being centrifuged. gs (gravities). Function of rpm and radius.

RPM Revolutions per minute. Speed of centrifugation. Determined by tachometer.

Radius Distance from center of rotation to bottom of tube when rotating. - -

Fixed-angle centrifuge Tubes are at fixed angle when rotating. Capable of higher speeds. Produces a slanted sediment surface.

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Horizontal centrifuge Tubes are in horizontal position when rotating. Produces a flat sediment surface.

Ultra centrifuge High-speed. Capable of 100,000 rpm.

Usual gs and time for 1000-1200 x g for 10 5 5 minutes centrifuging blood

Safety Make sure centrifuge is balanced. Don't open while spinning. Keep tubes capped. Clean daily with disinfectant.

Quality control Check speed every 3 months. Should be within 5 % . Check timer with stopwatch every 3 months. Should not be more than 10% off. Check temperatures of refrigerated centrifuges monthly. Should be within f Z°C. Check brushes every 2 months.


Packaging of Biologics for Shipment in Mail

Primary container Test tube, vial, etc. containing etiologic agent. Must be securely closed and watertight. Must be placed in a secondary container.

Secondary container Must be durable and watertight. Can contain more than one primary container, but not more than 50 mL total. Must contain enough absorbent material, such as paper towels, to absorb contents in case of breakage. Must be placed in an approved mailing container.

Mailing container Must be made of corrugated fiberboard, cardboard, wood, or other material of equivalent strength with an external dimension of no less than 3.9 inches

Labeling Etiologic agent label must be placed on mailing container. Must have biohazard symbol and phone number for CDC for notification in case of leakage.

(Regulations published in July 21, 1980 Federal Register under Interstate Shipment of Etiologic Agents)


v p e s of Glass

Aluminosilicate glass (Corex) Six times stronger than borosilicate and better able to resist clouding due to alkali and scratching.

Boron-free Alkali resistant. Poor heat resistance. Used for highly alkaline solutions.

Borosilicate glass (Kimax High resistance to thermal shock and chemical attack. Heavy walls to minimize and Pyrex) mechanical breakage. Most beakers, flasks, and pipets are made of this. Minimal

contamination of liquids by elements in the glass. Can be heated and autoclaved.

Flint glass Soda-lime glass containing oxides of sodium, silicon, and calcium. Least expensive but poor resistance to high temperatures and sudden changes of temperature. Resistance to chemical attack is only fair. Can release alkali and affect some determinations. Used for some disposable laboratory glassware.

High silica Heat, chemical, and electrical tolerance. Excellent optical properties. Used for high-precision analytic work, optical reflectors, mirrors.

Low actinic Amber or red. Used to reduce exposure to light.


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Glassware Inscriptions

A Class A. Meets high standards for accuracy. -

20°C Temperature of calibration. This is the temperature the glassware and solution should be for maximum accul acy.

TC To contain. The vessel is calibrated to hold a specified volume (e.g., volumetric flasks).

TD To deliver. The vessel is calibrated to deliver a specified volume (e.g., graduated cylinders).

Volumetric Glassware

Beaker Wide-mouthed, straight-sided jar with a pouring spout. Not accurate enough for critical measurements.

Erlenmeyer flask Sloping sides. Graduated markings. May be used to hold liquids, mix solutions, or measure noncritical volumes.

Florence flask Spherical base with a long cylindrical neck. Single calibration mark. Only for noncritical measurements.

Volumetric flask Pear-shaped. Long neck with a single calibration mark. Manufactured to strict standards. Glassware and solutions should be at room temperature. Used to accurately prepare standards and reagents. Should not be used to store solutions.

Graduated cylinder Upright, straight-sided tube with a flared base to provide stability. Used to make noncritical (graduates) measurements. Most are marked "TD." Should not be used for measuring volumes less than 5

mL or volumes less than 10% of capacity. Use the graduate closest in size to the volume to be measured.


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Glasd Pipets

Vblumetric pipet Ransfer pipet. Single calibration mark. Calibrated to accurately deliver a fixed volume of aqueous solutions such as nonviscous samples and standards. The last drop is touched off against the wall of the receiving vessel.

Ostwald-Folin pipet Transfer pipet. Similar to volumetric pipet but bulb is closer to the delivery tip, reducing the surface area in contact with the liquid. Etched ring near the upper end signifies that it is a blow-out pipet. Used for accurate measurement of viscous fluids such as whole blood or serum. Not widely used in laboratories today.

Serologic pipet Graduated or measuring pipet. Graduation marks down to the tip. Etched ring near the upper end signifies that it is a blow-out pipet. Used for preparing serial dilutions and measuring reagents. Generally not considered accurate enough for measuring samples or standards.

Mohr pipet Graduated or measuring pipet. Does not have graduation marks all the way to the tip and does not have a frosted band near the upper end. Delivery is made "point to point." Not commonly used.

-

Micropipets Disposable pipets for volumes ranging from 1-1000 ILL. Single calibration mark. Filled by r.

capillary action. TC. Must be rinsed out with diluent to deliver exact amount.


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'Ifrpes of Plastic

Polycarbonate Stronger than polypropylene and better temperature tolerance, but chemical resistance is not as good. Clear. Resistant to shattering. Used for centrifuge tubes.

Polyolefins Relatively inert chemically. Resistant to most acids, alkalis, and salts. (Concentrated sulfuric (polyethylene, acid attacks polyethylene). Polyethylene used in most disposable plasticware; can't be polypropylene) sterilized. Polypropylene can be sterilized.

Polyvinyl chloride Soft and flexible, but porous. Frequently used as tubing.

Teflon Extremely inert. Excellent temperature tolerance and chemical resistance. Nonwettable surface. Antiadhesive properties. Used for stir bars, stopcocks, tubing.


a

Grades of Chemicals

Reagent or analytical reagent grade Meets specifications of the American Chemical Society. Very high purity. Recommended for both qualitative and quantitative analysis.

Ultra pure reagents Also called spectrograde, nanograde, or high-performance liquid chromatography (HPLC) pure. Used for gas chromatography, HPLC, fluorometry, and trace metal determinations.

Chemically pure Not of sufficient purity to use as analytic reagents

Purified, practical, technical, or Not of sufficient purity to use as analytic reagents commercial nrade

USP or NF Grade Meet specifications of U.S. Pharmacopeia or National Formulary. Not injuri- ous to health. Not necessarily of sufficient purity to use as analytic reagents.


Reagent Grade Water

Type I Used in all quantitative and most qualitative lab procedures, for electrophoretic analyses, toxicology screening procedures, and in preparation of buffers, standards, and controls. Should not be stored.

'Ifrpe I1 Used in reagents with preservatives or reagents that are sterilized, and in most stains. Can be stored for short periods.

'Ifrpe I11 Used for washing and preliminary rinsing of glassware or as a source of water for further purification.


Water Purification

Process Removes Comments

Distillation Water is boiled and steam is Bacteria, pyrogens, particulate Does not meet conductivity condensed. matter, dissolved ionized requirement of type I water

solids, some dissolved organic contaminants

Deionization Water is passed through a Dissolved ionized gases and Resin must be frequently bed of mixed cation and solids replaced or regenerated. anion exchange resins.

Reverse osmosis Water is forced under Dissolved organic contaminants, Inadequate alone for produc- pressure through a semiper- ionic solids, bacteria tion of reagent grade water. meable membrane. Frequently used to pretreat

water before deionization.

Activated Water is passed through a Dissolved organic compounds Used with deionization. carbon filtration bed of activated carbon. and chlorine

Ultrafiltration Water is passed through Particulate matter, bacteria, semipermeable membranes pyrogens of pore size 10.22 pm.

UItraviolet Exposure to 185 nm for Organic contaminants Used after other purification oxidation and oxidation; 254 nm for (oxidation) and bacteria processes. sterilization sterilization (sterilization)


Brightfield Microscopy Fast Facts

Achromatic objective Least expensive objective. Partially corrects for chromatic and spherical aberrations.

Aperture diaphragm Controls angle and amount of light sent to objective

Binocular microscope One with two oculars

Blue filter Used to eliminate yellow color emitted by tungsten

Brightfield microscope Uses transmitted light and lenses. Objects appear dark against white background. Used for most routine clinical work.

Compound microscope One with two lens systems-objectives and oculars

Condenser Focuses light on specimen

Depth of focus Distance throughout which all parts of specimen are clearly in focus simultaneously

Field diaphragm Limits area of illumination to image field

Field of view Area of specimen that can be seen

Immersion oil Used to help objective gather light from a wide numerical aperture. Provides high resolution. Type B (high viscosity) is commonly used.

Kohler illumination Method of focusing and centering light path and spreading light uniformly. Ensures opti- mum contrast and resolution.

Magnification, total Magnification of the ocular x magnification of the objective. lOOOX is the highest magnification achievable with a brightfield microscope.

Continued


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Brightfield Microscopy Fast Facts

Numerical aperture (NA) Mathematical expression of light admitted by lens. The higher the NA, the greater the resolution.

Objectives Lenses attached to the revolving nosepiece. Most commonly used are low power (10x1, high power (40X), and oil immersion (50 or 1 0 0 ~ ) .

Ocular Eye piece. Usually lox.

Parcentric Property whereby an object in the center of a field at one magnification remains in the center of the field at other magnifications

Parfocal Property whereby an object remains in focus from one magnification to another

Planachromatic objective More expensive objective that corrects for curvature of the field, resulting in a flat field with uniform focus

Resolution Ability to reveal fine detail and distinguish between two close points - -

Rheostat Light control knob. Light intensity should not be regulated by the condenser or di- aphragms.

Tbngsten-halogen bulb Type of bulb used for brightfield microscopy

Virtual image - - -

Image seen through the microscope. It is upside down and reversed.

Working distance Distance between the slide and the objective. Decreases with higher magnification objectives.


L

r

Other 'Ifrpes of Microscopy

Explanation Application in Clinical Laboratory

Light Microscopes

Darkfield Brightfield microscope with special condenser. Objects Identification of live Deponema appear white against a black background. pallidum and other microorganisms

Fluorescent Brightfield microscope with two special filters. Excitation filter Direct and indirect fluorescent selects wavelength of light presented to specimen. Barrier or antibody stains in microbiology emission filter permits specific wavelength of fluorescent light and immunology from specimen to pass to eyepiece. Fluorescent dyes absorb light of one wavelength and emit light of a longer wavelength. Objects appear green, yellow, or orange against a black background.

Interference Brightfield microscope with special slit aperture below Wet mounts contrast condenser, polarizer, and special amplitude filter (modulator)

in back of each objective. Gives a 3-D effect to unstained specimens.

Phase Brightfield microscope with phase condenser and phase Manual platelet counts, urine contrast objectives. Subtle differences in refractive index are converted sediments (good for hyaline casts)

into clear-cut variations of light intensity and contrast. Good for living cells, unstained specimens.

Continued

1 32 CLINICAL LABORATORY PRACTICE REVIEW

i

Other IIfrpes of Microscopy

Explanation Application in Clinical Laboratory

Polarizing Brightfield microscope with two crossing filters-polarizing Identification of crystals in urine and filter located below condenser, analyzer between objective synovial fluid. Confirmation of fat or and eyepiece. Objects that can refract light (birefringent) oval fat bodies in urine sediment. appear white against a black background.

Electron Microscopes

Transmission Beam of electrons passes through specimen and is focused Virology, cells (organelles) onto a fluorescent screen or photographic plate. Magnification >100,00OX.

Scanning Ream of electrons strikes surface of specimen and is focused Virology, cells (surface) onto photographic film or cathode ray tube. Forms 3-D image. Magnification >1,00OX.

CLINICAL LABORATORY PRACTICE REVIEW 33 ! i

Computers - - - - -- - - - --

Hardware The physical parts of a combuter

CPU Central processing unit. The electronic portion of a computer.

Input devices Mechanisms whereby the computer receives data; e.g., keyboards, bar-code readers, computer links (interfaces)

Output devices Means by which computer delivers information; e.g., printers, CRT screens

RAM Random access memory. "Volatile memory." Working memory used for temporary storage of programs and data. Content is lost each time the computer is turned off.

ROM Read only memory. Part of the memory that js permanently protected from being modified, erased, or written over. Not affected by loss of power. Used for boot-level and other system instructions.

Hard drive Magnetic-coated metal plate inside the CPU for storing data

Floppy drive Removable flexible plastic disk with a magnetic coating, used to store information external to the CPU

Optical disk Laser-read compact disk

Disk drive Unit that allows data to be extracted and entered on disks

Software Programs that tell the computer what to do

Systems software Programs that control and run the operating system of the computer

Application software Programs designed to meet specific needs of the users

Continued

34 CLINICAL 1,ABORATORY PRACTICE REVIEW

Computers

Word processor Application program that allows for the manipulation of text. Used to write letters, reports.

Spreadsheet Application program used to manipulate numbers and perform mathematical calculations. Used to prepare financial statements, budgets, cash projections.

Database Application program used to organize, store, sort, and retrieve data (words or numbers).


. - _-a ', "

Computer Networks

Local area network (LAN) A computer network that connects computers and their equipment in close geographic proximity (e.g., building, campus)

Wide area network (WAN) A computer network that connects computers and their equipment over a large geographic area (e.g., Internet)

Laboratory information system (LIS) Network of computer components designed to incorporate all aspects of the informational needs of the laboratory and its customers from the intake of requests for services to the delivery of results. Can provide patient information, test information, work lists, test results, financial functions, pro- ductivity and workload monitoring, quality management, interface with other computer systems.

Client server A system that allows users to tap into the LIS and extract the information they want.

i 1 36 CLINICAL LABORATORY PRACTICE REVIEW

Phases of Quality Assurance

Phase Definition Examples - - - - - - - - - - -

Preanalytical Everything that precedes actual test performance Test ordering by physician, test ordering by nursing/clerical staff, patient preparation, patient identification, specimen collection, specimen transport, specimen processing

Analytical Everything related to the actual testing process Test analysis, quality control, reagents, cali. bration, preventive maintenance

Postanalytical Everything that comes after test analysis Verification of calculations and reference ranges, review of results, procedures for notification of critical values, result reporting, test interpretation by physician, follow-up patient care


t d

Quality Assurance

Quality assurance Process by which a laboratory ensures quality results by closely monitoring the preanalytical, analytical, and postanalytical stages of testing.

Quality control Part of the analytical stage of quality assurance; process of monitoring results from control samples to verify quality of patient results.

Sensitivity Ability of the method to detect slight differences in concentration. High sensitivity means few false negatives and is desirable in screening tests.

Specificity The ability of the method to determine solely the compound it is supposed to measure. High specificity means few false positives and is desirable in confirmatory tests.

Accuracy How close a measurement is to the true value. Implies freedom from error.

Precision Reproducibility. Indicates how close test measurements are to each other when the same test is run on the same sample time after time. Usually expressed in terms of standard deviation (SD) or coefficient of variation. Precision does not imply accuracy. Precision implies freedom from variation.

Imprecision A large amount of scatter about the mean. Most often caused by technique errors such as variability in pipetting or inattention to detail by the operator. Results in an increase in the SD or a broadening of the distribution about the mean.

Standard A solution that contains a known amount of an analyte. Used to calibrate an assay method.

Standard, primary A substance that can be accurately weighed or measured to produce a solution of an exactly known concentration. Must be 99.98% pure. This level of purification is impossible to attain with most biologic materials and is not necessary for routine testing.

Continued


Quality Assurance Continued

Coefficient of Expresses standard deviation as a percentage. CV % = [SD + mean) X 100. Allows variation (CV) comparison of the precision of tests having different concentration ranges or units. The

lower the CV, the higher the precision.

Normal distribution The Gaussian bell-shaped curve. Mean + 1 SD = 68.2 % of observations Mean + 2 SD = 95.45 % of observations (referred to as 95 % confidence interval] 1 Mean * 3 SD = 99.73% of observations

Levey-Jennings chart Normal distribution curve lying on its side, marked with the mean and *1, 2, and 3 SD. Thirty sequential control results are plotted on x axis.

Control limits Numerical limits within which the values of controls must fall for the assay to be considered valid. Many labs use the mean ?2 SD. One determination in 20 will fall outside 2 2 SD. This is an anticipated part of normal variation.

Outlier A control result outside of the established limits. May be due to chance or may indicate a problem in the test system. If it occurs more frequently than once in 20 successive runs, then patient results cannot be reported. Investigation must be carried out.

Shift Six consecutive control values fall on the same side of the mean.

Trend Control values increasing or decreasing for six consecutive runs.

Error, random Error affecting a particular determination that does not recur in a regular pattern (e.g., error due to use of dirty glassware or the wrong pipet, voltage fluctuation, sampling error, anticoagulant or drug interference]. Indicated by a control value that is significantly different from the other values on a Levey-Jennings chart, or violation of the I,, and R4, Westgard rules. Usually a one-time error and controls and samples can be rerun with success.

Continued 40 CLINICAL LABORATORY PRACTICE REVIEW


Procedure manual A set of instructions for methods used in the laboratory. Must be approved, signed, and dated by the director of the laboratory.

Proficiency testing Testing of unknowns submitted by an outside agency. Unknowns are not to receive prefer- ential treatment. Results are reported to the agency, which compares them to results from other laboratories. One commonly used provider of proficiency testing is the College of American Pathologists. CLIA '88 requires that all laboratories performing moderately or highly complex tests participate in proficiency testing.

Correlation study A study to verify the accuracy of a new method, where split patient samples are analyzed by the existing method and the new method. Requires a minimum of 40 patient samples representing a wide range of concentrations. Reference values (existing method) are plotted on the x axis and new values on the y axis. Perfect correlation is indicated by a straight line passing through zero at a 45" angle. The correlation coefficient (r) can be derived mathemat- ically. Values range from - 1 to + 1. A value of 0 indicates no correlation between the two methods. A value of + 1 indicates a perfect direct relationship between the two methods. A value of 0.95 or higher is considered excellent agreement.


Westgard Multirules

Rule Explanation m e of Error Comments

l,, One control exceeds the mean by more than 2 SD, but less than 3 SD. A warning flag that indicates a possible change in accuracy or precision.

Initiates testing of other rules. If there are no violations of the remaining rules, the control values are considered acceptable.

l,, One control exceeds the mean by more Random Rejection rule than 3 SD.

2,s l b o consecutive controls exceed the mean Systematic Rejection rule by more than 2 SD, but less than 3 SD and in the same direction.

R,, The difference between two consecutive Random Rejection rule controls is greater than 4 SD.

4,s Four consecutive controls exceed the mean Systematic Rejection rule by more than 1 SD and in the same direction.

10, Ten consecutive controls fall on one side of Systematic Rejection rule the mean.


b 2

A Generic Approach to an Out-Of-Range Control

Step Rationale

Repeat the same control. Check for human error. V a h rn-Qht have been due to expected random error. (1 in 20 results will be outside ?2 SD.)

Run a new vial of the same control. First vial may have been outdated, improperly stored, contaminated.

Run a new control from a different lot. First lot may have been outdated, improperly stored.

Perform maintenance, check reagents, Problem might be due to inadequate preventive maintenance. Reagents and rerun the control. might have been outdated, improperly stored, contaminated.

Recalibrate and rerun the control. Calibration may have shifted.

Get assistance. Supervisor or service rep might be able to locate source of problem.

Document corrective action. Provides a record for future reference. Will point out repetitive problems.


Miscellaneous Quality Control

Balances Verify accuracy monthly using class S weights.

Diluters Verify accuracy and precision on receipt, after service or repair, and on a regular schedule using spectrophotometric procedure with potassium dichromate.

Dispensers Verify accuracy and precision on receipt, after service or repair, and on a regular schedule using gravimetric method.

Micropipets Verify accuracy and precision on receipt, after service or repair, and on a regular schedule. Primary method for calibration is gravimetric method. Secondary method is spectrophotometric procedure with potassium dichromate (unacceptable for volumes <10 $1 -

Temperature-controlled Check daily with a National Institute for Standards and Technology (N1ST)-certified equipment: water thermometer or one calibrated against an NIST-certified thermometer. Most should be baths, heat blocks, within t 1°C. refrigerators, freezers

Thermometers Verify every 6-12 months with an NIST-certified thermometer. Should be within 21°C.

Water Monitoring of resistivity (ionic content), microbiologic content, pH, soluble silica.


Procedure Manual Format (NCCLS and CLIA '88)

1. Requirements for specimen collection and processing and criteria for specimen rejection

2. Procedures for microscopic examinations, including the detection of inadequately prepared slides

3. Step-by-step performance of the procedure, including test calculations and interpretation of results

4. Preparation of slides, solutions, calibrators, controls, reagents, stains, and other materials used in testing

5. Calibration and calibration-verification procedures

6. Reportable range for patient test results

7. Control procedures

8. Remedial action to be taken when calibration or control results fail to meet the laboratory's criteria for acceptability

9. Limitations in methodologies, including interfering substances

10. Reference range

11. Imminent life-threatening laboratory results (panic or critical vaIues)

12. Pertinent literature references

13. Appropriate criteria for specimen storage and preservation to ensure specimen integrity until testing is completed

14. The laboratory's system for reporting patient results, including the protocol for reporting critical values

15. Description of the course of action to be taken in the event a test system becomes inoperable

16. Criteria for the referral of specimens, including procedures for specimen submission and handling

Manufacturer's package inserts may be used for items 1-13. Items not provided by manufacturer must be provided by the laboratory.

Procedures and changes must be approved, signed, and dated by the director. Procedures must be retained for 2 years after date of discontinuation.


Col .lege of American Pathologists (CAP) Guidelines for Retention of Specimens

Specimen Retention Period - - - - - - -

Blood bank specimens 7 days post-transfusion or 10 days post-crossmatch - -- -

Blood smears 7 days

Bone marrow smears 10 years - Microbiology slides, stained 7 days

Serum/CSF/body fluids 24 hours

CLlNICAL LABORATORY PRACTICE REVIEW 47

CAP Guidelines for Retention of Records

Record n p e Retention Period

Accession records 2 years

Donor and recipient records Indefinitely

Instrument maintenance records 2 years

Patient test records 2 years -- -- Quality control records 2 years (exception: 5 years for blood bank)


-

Quality Assurance

Quality assurance Process by which a laboratory ensures quality results by closely monitoring the preanalytical, analytical, and postanalytical stages of testing.

Quality control Part of the analytical stage of quality assurance; process of monitoring results from control samples to verify quality of patient results.

Sensitivity Ability of the method to detect slight differences in concentration. High sensitivity means few false negatives and is desirable in screening tests.

-

Specificity The ability of the method to determine solely the compound it is supposed to measure. High specificity means few false positives and is desirable in confirmatory tests.

Accuracy How close a measurement is to the true value. Implies freedom from error. -- - -

Precision Reproducibility. Indicates how close test measurements are to each other when the same test is run on the same sample time after time. Usually expressed in terms of standard deviation (SD) or coefficient of variation. Precision does not imply accuracy. Precision implies freedom from variation.

Imprecision A large amount of scatter about the mean. Most often caused by technique errors such as variability in pipetting or inattention to detail by the operator. Results in an increase in the SD or a broadening of the distribution about the mean.

Standard A solution that contains a known amount of an analyte. Used to calibrate an assay method. I

Standard, primary A substance that can be accurately weighed or measured to produce a solution of an exactly known concentration. Must be 99.98% pure. This level of purification is impossible to attain with most biologic materials and is not necessary for routine testing.

- - - - -


Continued

Quality Assurance Continued - - - - - - - - - -

Coefficient of Expresses standard deviation as a percentage. CV % = (SD + mean) X 100. Allows variation (CV) comparison of the precision of tests having different concentration ranges or units. The

lower the CV, the higher the precision.

Normal distribution The Gaussian bell-shaped curve. Mean 5 1 SD = 68.2 % of observations Mean 2 2 SD = 95.45% of observations (referred to as 95% confidence interval) Mean 2 3 SD = 99.73% of observations

Levey-Jennings chart Normal distribution curve lying on its side, marked with the mean and ?1, 2, and 3 SD. Thirty sequential control results are plotted on x axis.

Control limits Numerical limits within which the values of controls must fall for the assay to be considered valid. Many labs use the mean 5 2 SD. One determination in 20 will fall outside 2 2 SD. This is an anticipated part of normal variation.

Outlier A control result outside of the established limits. May be due to chance or may indicate a problem in the test system. If it occurs more frequently than once in 20 successive runs, then patient results cannot be reported. Investigation must be carried out.

Shift Six consecutive control values fall on the same side of the mean.

Trend Control values increasing or decreasing for six consecutive runs.

Error, random Error affecting a particular determination that does not recur in a regular pattern (e.g., error due to use of dirty glassware or the wrong pipet, voltage fluctuation, sampling error, anticoagulant or drug interference). Indicated by a control value that is significantly different from the other values on a Levey-Jennings chart, or violation of the l,, and R,, Westgard rules. Usually a one-time error and controls and samples can be rerun with success.

Continued 40 CLINICAL LABORATORY PRACTICE REVIEW


Standard, secondary A substance of lower purity whose concentration is determined by assay and comparison with a primary standard.

Calibrator Substance with an assigned value that the manufacturer establishes by using a reference method or by using reference materials traceable to primary standards. Used to set the value reported by the laboratory's method or instrument.

Control A sample that is chemically and physically similar to the unknown specimen and is tested in exactly the same manner. Monitors precision of the test system. Controls should be run at all levels of clinical importance. For quantitative tests, CLIA '88 requires that at least two control samples of different concentrations (normal and abnormal) be assayed at least every 24 hours. Some states mandate three pools (low abnormal, normal, high abnormal) for some tests. [For qualitative tests, positive and negative controls must be included with each run of patient specimens.)

- -

Measures of Statistical parameters describing the spread of data about the mean; e.g., standard dispersion deviation, coefficient of variation, and range. Measurements of precision.

Range - - - - - - - - - - - - - - - - -

Difference between the highest and lowest values in a data set. An indication of the dispersion of data points.

-

Mean Sum of all observations divided by the number of observations. The average of all observations.

Standard deviation Statistical expression of the dispersion of values around the mean. Equal to the square root (SD) of the sum of the squared differences from the mean divided by the number of values minus

one. A minimum of 20 analyses are needed for the calculation.

Continued

CLINICAL LABORATORY PRACTlCE REVIEW 39


Error, systematic Recurring, measurable error that is inherent in a test procedure. Is continuous and affects all results. Indicated by a trend or a shift on a Levey-Jennings chart, or violation of the 2,,, 4,,, or 10, Westgard rules. Requires investigation to determine cause. Examples: dirty photome- ter, faulty ISE, evaporation or contamination of standards or reagents.

False rejection Rejection of an analytical run because quality control results indicate arlalytical problems that are not really present. Use of Westgard rules minimizes false rejections.

Linearity Reportable range.

Reportable range The range of concentrations that may be reported without the need for further treatments such as dilution. Also known as linearity.

Reference range Formerly called normal values. Each laboratory should set its own reference ranges for all analytes. 100-150 data points should be gathered from a representative healthy client population. Data are arranged in sequential order and the data points that occupy the 2.5 % position at the low end and the 97.5% position at the high end define the range. (95% confidence interval. One normal person in 20 will fall outside the reference range.)

Significant change A change greater than three times the quality control SD. Indicates a real change in the patient.

Delta check Comparison of patient data with previous results. Delta limits are set to detect specimen mix-up or other errors. When limits are exceeded, one must determine if a major medical change has occurred in the patient or if a laboratory error occurred.

Preventive A schedule of daily, weekly, and monthly maintenance of equipment designed to keep it in maintenance peak operating condition. Must be documented.

Continued


Commonly Used Prefixes in the Metric System Deci = 10-1

Centi = 10-2

Milli = 10--3

Micro = 10-6

Nano = 10-9

Pico = 10-12

Femto = 10-15


d

General Laboratory Calculations

If the temperature of a refrigerator is 4"C, what is the temperature in OF?

O F = (1.8 x C) + 32 = 39.2 If the room temperature is 73"F, what is the temperature in "C?

73 = 1.8C + 32

J Z 6 - x ) ~ Standard Deviation (SD) = - Z = sum, x = individual value, K = mean, n = number of values

Coefficient of variation (%) = SD x 100 What is the CV for a procedure whose mean is 100 and whose Mean standard deviation is 3?

cv = 3 x 100 = 3% 100

Dilution = What is the dilution if 0.1 mL of serum is diluted with 0.4 mL of saline? Vol. of specimen 0.1 - 0.1 1

- Vol. of specimen + vol. of dilluent 0.1 + 0.4 0.5 5

How would you prepare a 1:10 dilution of urine? 1 part of urine + 9 parts of diluent

Correcting for a dilution: Value obtained A specimen for a glucose is diluted 1:s. The value of the diluted for diluted specimen x reciprocal of specimen is 100 mg/dL. What value should be reported? dilution 100 mg/dL x 5 = 500 mg/dL


C

Laboratory Tests/Medical Records t I Laboratory tests can only be ordered by physicians.

Requests for laboratory tests must be made in writing or by electronic means.

Test results are not to be released to anyone except the treating physician without the patient's consent.

Unauthorized release of test results can lead to charges of breach of confidentiality or invasion of privacy.

The most common cause of erroneous lab reports is clerical error.

To correct an erroneous report, draw a line through the incorrect value so that it is still legible. Do not erase or white-out. Record the corrected value and date the entry.

Laboratory records are the property of the laboratory or of the hospital if the laboratory is owned by the hospital.

Patients have a right to their own healthcare information, including laboratory test results.


i I

SECTION 2

Clinical Chemistry Review

Conventional Units vs. SI Units Comparison of Selected Reference Ranges

Amlyte Conventional Units sz Units

Bilirubin, total 0.2-1.0 mg/dL 3-1 7 pmol/L

BUN 7- 18 mg/dL 2.5-6.4 mmol/L

Calcium, total 8.7-10.2 mg/dL 2.18-2.25 mmol/L

Chloride 98-106 mEq/L 98-106 rnmol/L -- ~ - - - --

Creatinine Male: 0.6-1.2 mg/dL Male: 53-106 mmol/L Female: 0.5-1.1 mg/dL Female: 44-97 mmol/L

Glucose, fasting 70-110 mg/dL 3.9-6.0 mmol/L

Magnesium 1.6-2.4 mEq/L 0.65-1.0 mmol/L

Potassium 3.4-5.0 mEq/L 3.4-5.0 mmol/L

Sodium

Total protein 6.5-8.3 g/dL 65-83 g/L

Uric acid Male: 3.5-7.2 mg/dL Female: 2.6-6.0 mg/dL

Male: 208-428 pmol/L Female: 155-357 pmol/L

54 CLINICAL CHEMISTRY REVIEW

Patient Variables That Affect Chemistry Values

Factor Examples of Amlytes Affected

Age Albumin, alkaline phosphatase, phosphorus, cholesterol

Gender Albumin, alkaline phosphatase, creatinine, uric acid

Diurnal variation 1' in AM: adrenocorticotropic hormone (ACTH), cortisol, iron '? in PM: acid phosphatase, growth hormone, parathyroid hormone (PTH), thyroid-

stimulating hormone (TSH)

Day-to-day variation 20% or more for alanine aminotransferase (ALT), bilirubin, iron, TSH, triglycerides

Recent food ingestion 1' glucose, insulin, triglycerides, gastrin .1 chloride, phosphorus, potassium

Foods and beverages Avocados, bananas, eggplant, pineapple, plums, and walnuts cause false-positive 5-hydroxyindole acetic acid.

Bananas, vanilla cause false-positive vanillylmandelic acid (VMA]. Caffeine increases cortisol. Alcohol increases triglycerides and gamma glutamyl transaminase (GGT).

Posture '? when standing: albumin, cholesterol

Activity - - -

'? in ambulatory patients: creatinine kinase (CK) '? with exercise: lactic acid, creatinine, protein, CK, aspartate aminotransferase (AST],

lactate dehydrogenase (LD) .1 with exercise: cholesterol, triglycerides

Stress 1' ACTH, cortisol, catecholamines

CLINICAL CHEMISTRY REVIEW 55

Effect of Anticoagulant Contamination on Chemistry Analytes

Anticoagulant Amlytes Affected

Ethylenediaminetetra- calcium, magnesium acetic acid (EDTA) Inhibits alkaline phosphatase, CK, amylase

? potassium with K, EDTA, ? sodium with Na, EDTA.

Sodium fluoride/ .1 calcium, magnesium potassium oxalate Fluoride inhibits enzymes; interferes with BUN by urease and uric acid by uricase

Sodium citrate .1 calcium, magnesium Inhibits alkaline phosphatase, CK, amylase

Heparin Inhibits alkaline phosphatase ? ammonia with ammonium heparin, ? lithium with lithium heparin, ? sodium with

sodium heparin if tube is not completely filled

Lithium iodoacetate Inhibits CK ? lithium


Collection, Processing, and Storage Factors That Affect Chemistry Results

Factor Examples of A ~ l y t e s Affected

Squeezing site of capillary puncture I' potassium

Pumping fist during venipuncture '? potassium, lactic acid, calcium, phosphorus -1 PH

Tourniquet > 1 minute ? potassium, total protein, cholesterol, iron

IV fluid contamination Common cause of I' glucose, potassium, or drugs, depending on IV

Serum separator tubes -1 tricyclic antidepressants, some antiarrhythmics

Hemolysis ? potassium, magnesium, phosphorus, LD, acid phosphatase, AST, iron, total protein, ammonia

Exposure to light -1 bilirubin, carotene

Delay in separating serum/plasma ? ammonia, lactic acid, potassium, magnesium, LD -1 glucose

Temperature of storage -1 at room temperature: glucose, acid phosphatase I' at room temperature: lactic acid, ammonia -1 at 4°C: LD


Factors That Affect Enzyme Reactions

Factor Effect Comments

Substrate First-order kinetics: [enzyme] > [substrate]. Use zero-order reaction [excess substrate). concentration Reaction rate proportional to [substrate].

Zero-order kinetics: [substrate] > [enzyme]. Reaction rate proportional to [enzyme].

Enzyme Velocity of reaction is proportional to [enzyme] Report in U/L. [U = International Unit = concentration as long as [substrate] > [enzyme]. amount of enzyme that will catalyze the

reaction of 1 micromole of substrate per minute under specified conditions.)

PH Extremes of pH may denature enzymes. Most reactions occur at pH 7-8. Use buffers.

Temperature An increase of 10°C doubles the rate of reaction 37°C is most commonly used in U.S. until around 40-50°C when denaturation of enzyme may occur.

Cofactors Activators = inorganic cofactors (metallic and Must be present in excess. nonmetallic ions) Common cofactors: nicotinamide adenine

Coenzymes = organic cofactors (nucleotide dinucleotide (NAD) o nicotinamide adenine phosphates and vitamins) dinucleotide, reduced form (NADH). NADH

The higher the concentration of cofactors, has absorbance at 340 nm; NAD does not. the greater the rate of reaction.

Inhibitors Interfere with the reaction. Must be lacking.


L

----.*..- &

Differences in Analyte Concentrations / 1

Higher in Higher in Higher in Higher in Plasma Serum Plasma Higher in Capillary Venom Blood than in than in than in Blood than in than in Serum Plasma Whole Blood Venom Blood Capillary Blood

Total protein Potassium Glucose Glucose (in postprandial Calcium LD Phosphorus specimen) Total protein Calcium Glucose Potassium

CK Bicarbonate Alkaline phosphatase

(ALP) Albumin Aspartate aminotransferase

(ASTI Triglycerides


L

Differences in Electrolyte Concentrations

Higher Concentration in RBCs Higher Concentration in Plasma

Potassium Sodium Phosphorus Chloride Magnesium


Chemistry Panels

Panel Tesl

Basic metabolic panel Sodium, potassium, chloride, CO,, glucose, creatinine, BUN

Comprehensive metabolic panel Sodium, potassium, chloride, CO,, glucose, creatinine, BUN, albumin, total protein, ALP, AST, bilirubin, calcium

Electrolyte panel Sodium, potassium, chloride, CO,

Hepatic function panel Albumin, alanine aminotransferase (ALT), AST, ALP, bilirubin (total and direct)

Lipid panel - -

Cholesterol (total), HDL cholesterol, triglycerides

Thyroid panel Thyroxine (T,), thyroid hormone binding ratio (THBR or T3 uptake), thyroid- stimulating hormone (TSH)


..~.".~_Tlln..-sr

Chemistry Tests

Analyte Reference Range Clinical Significance Other

Sodium 138-146 mmol/L T (hypernatremia): hyperaldosteronism, or mEq/L ingestion or administration of large

amounts of sodium, excessive sweating, burns, hyperventilation, diabetes insipidus, osmotic diuresis .1 (hyponatremia): heart failure, liver disease, nephrotic syndrome, renal failure, inappropriate antidiuretic hormone (ADH) secretion, excessive fluid intake, vomiting, diarrhea, burns, diuretic therapy, hypoaldosteronism

Potassium 3.7-5.3 mmol/L '? (hyperkalemia): acidosis, crush injuries, or mEq/L tissue hypoxia, insulin deficiency, digitalis

overdose, IV administration, transfusion of aged blood, renal failure, hypoaldosteronism, diuretics, defects in renal tubular secretion .1 (hypokalemia): alkalosis, increased insulin, diuretics, decreased intake, increased GI or urinary loss

Major extracellular cation. Contri- butes almost half to plasma osmolality. Central role in maintain- ing normal distribution of water and osmotic pressure. Levels regulated by aldosterone. Ion-selective electrode is most common method. Artifac- tually low levels in samples with elevated lipids or protein with some methods. Normal Na+:K+ ratio in serum approximately 30:l. Critical: < 120 and > 160. If sodium heparin is used, tubes must be completely filled.

Major intracellular cation. Critical: <2.8 and X . 2 . Hyper- and hypokalemia cause cardiac arrhyth- mias. Pseudohyperkalemia due to squeezing site of capillary puncture, prolonged tourniquet time, pumping of fist during venipuncture, contamination with IV fluid, hemolysis, prolonged contact with RBCs, icing specimen before separation, leukocytosis, thrombocytosis. Serum

Continued 62 CLINICAL CHEMISTRY REVIEW

F - w

Chemistry Tests Continued


values 0.1-0.2 mmol/L higher than plasma due to release from platelets during clotting. Most common method is ISE with membrane in- corporating valinomycin.

Chloride 98-109 mmol/L T prolonged diarrhea, renal tubular disease, hyperparathyroidism, dehydration J prolonged vomiting, burns, salt-losing renal diseases, overhydration

CO,, total 23-30 mmol/L ? metabolic alkalosis, compensated respiratory acidosis

J metabolic acidosis, compensated respiratory alkalosis

Major extracellular ion. Maintains hydration, osmotic pressure, ionic balance. Changes usually parallel changes in sodium. Most common method is ISE. Other methods: colorimetric and coulometric titration. - -

Primarily bicarbonate. Keep sample capped to prevent loss of CO,.

Glucose, fasting

70-110 mg/dL ? (hyperglycemia): diabetes mellitus, Major source of cellular energy. (3.9-6.1 mmol/L) pancreatitis, hepatic failure, renal disease Levels decrease at room tempera-

J (hypoglycemia): insulinoma, insulin- ture. Use sodium fluoride or iodo- induced hypoglycemia, acetate to preserve. Hexokinase is hypopituitarism most specific and widely used

method. Critical: <40 mg/dL

Continued


p- - -?Zb.-- --- Chemistry Tests Continued


(coma, seizures, death) and >SO0 mg/dL (acidosis, coma). > 126 mg/dL on more than one occasion = diabetes mellitus.

BUN 8-26 mg/dL T kidney disease Synthesized by liver from ammonia. (3.0-9.2 mmol/L) -1 overhydration or liver disease Excreted by kidneys. Urease reagent.

Do not use sodium fluoride, EDTA, citrate, or ammonium heparin. Test is not sensitive. Urine-dilute 1:20 or 150 and refrigerate or acidify.

Creatinine 0.7-1.5 mg/dL T kidney disease 160-130 pmol/L)

Waste product formed by dehydration of creatine (mainly in muscles). Jaffe's reaction (alkaline picrate) is nonspecific. Enzymatic methods are more specific. Tests are not sensitive. Normal BUN:creatinine ratio = 12-20. Urine-dilute 1 :loo.

Uric acid 2.5-7.0 mg/dL T gout, renal failure, ketoacidosis, Increased = risk of renal calculi and (0.15-0.40 lactate excess, high nucleoprotein diet, joint tophi.. Uricase method. EDTA mmol/L) leukemia, lymphoma, polycythemia and fluoride interfere. Urine-adjust

-1 administration of ACTH, renal pH to 7.5-8 to prevent precipitation. tubular defects

Continued


- P. 4'

Chemistry Tests Continued

Amlyte Reference Range Clinical Significance Other

Cholesterol Desirable T hypothyroidism, nephrosis, diabetes, Direct correlation with risk of coronary <200 mg/dL liver disease artery disease.

severe liver insufficiency

Mglycerides Desirable T risk factor for atherosclerotic cardio- Main form of lipid storage. Requires <200 mg/dL vascular disease a fasting specimen.

k malnutrition

Total protein 6.0-8.0 g/dL T dehydration, chronic inflammation, <4.5 g/dL associated with peripheral (60-80 g/L) multiple myeloma edema. Biuret method. Alkaline

-1 nephrotic syndrome, malabsorption, copper reagent reacts with peptide overhydration, hepatic insufficiency, bonds. malnutrition, agammaglobulinemia

Albumin 3.5-5.0 g/dL T dehydration Largest fraction of plasma proteins. (35-50 g/L) -1 malnutrition, liver disease, Synthesized by liver. Regulates

nephrotic syndrome, osmotic pressure. Measure by dye chronic inflammation binding; e.g., bromcresol green(BCG),

bromcresol purple (BCP) . Bilirubin, 0.1-1.2 mg/dL T liver disease, hemolysis, hemolytic From breakdown of RBCs. Metabolized total (1.7-20.5 pmol/L) disease of the newborn in liver. In infants, >20 mg/dL associated

with brain damage (kernicterus) and may require exchange transfusion. Protect from light. Diazo reagent + methyl alcohol or caffeine.

Continued CLINICAL CHEMISTRY REVIEW 65

Contents SECTION 1: CLINICAL LABORATORY PRACTICE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . .

1 . Credential~ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 . Regulatory Agencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 . Agencies that Issue Guidelines/Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 . Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 . OSHAStandards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6 . Bloodborile Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7 . Hazard Categories of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8 . Hazard Identification System-National Fire Protection Association (NFPA) . . . . . . . . . . . . . . . . . . . . . . . 10 9 . Storage of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

10 . Firesafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 11 . Commonly Used Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 12 . Tests Requiring a Fasting Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 13 . Recommended Order of Drawing/Filling Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 14 . Situations in Phlebotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 15 . Specimens Requiring Special Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 16 . SpecimenProcessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 17 . Exan~ples of Criteria for Specimen Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 18 . Centrifuge Fast Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 . Packaging of Biologics for Shipment in Mail 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 . TypesofGlass 22

21 . Glassware Inscriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 22 . Volumetric Glassware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 23 . GlassPipets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 . Types of Plastic 26 25 . GradesofChemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 . Reagent Grade Water 28 27 . Water Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Contents SECTION 1: CLINICAL LABORATORY PRACTICE REVIEW ...................................... 1

1 . Credentialing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 . Regulatory Agencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 . Agencies that Issue Guidelines/Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 4 . Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5 . OSHAStandards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 6 . Bloodborne Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 7 . Hazard Categories of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 8 . Hazard Identification System-National Fire Protection Association (NFPA) . . . . . . . . . . . . . . . . . . . . . . . 10 9 . Storage of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

10 . Firesafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 11 . Commonly Used Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 12 . Tests Requiring a Fasting Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 13 . Recommended Order of Drawing/Filling Thbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 14 . Situations in Phlebotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

. 15 Specimens Requiring Special Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 16 . Specimen Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 17 . Examples of Criteria for Specimen Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 18 . Centrifuge Fast Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 . Packaging of Biologics for Shipment in Mail 21 20 . TypesofGIass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 21 . Glassware Inscriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 22 . Volumetric Glassware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 23 . Glass Pipets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 24 . Types of Plastic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 . Grades of Chemicals 27

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 . Reagent Grade Water 28 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 . Waterpurification 29

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 . Brightfield Microscopy Fast Facts 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 . Other v p e s of Microscopy 3

30 . Computers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 . ComputerNetworks ?

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 . Phases of Quality Assurance 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 . Quality Assurance 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 . Westgard Multirules 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 . A Generic Approach to an Out-of-Range Control 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 . Miscellaneous Quality Control 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 . Procedure Manual Format (NCCLS and CLIA '88) 4 38 College of American Pathologists (CAP) Guidelines for Retention of Specimens . . . . . . . . . . . . . . . . . . . . . 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 . CAP Guidelines for Retention of Records 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 . Laboratory TestsIMedical Records 4

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 . Commonly Used Prefixes in the Metric System 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 . General Laboratory Calculations !

................................................ SECTION 2: CLINICAL CHEMISTRY REVIEW 5 43 . Conventional Units vs . SI Units-Comparison of Selected Reference Ranges . . . . . . . . . . . . . . . . . . . . . . . 5

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 . Patient Variables that Affect Chemistry Values 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 . Effect of Anticoagulant Contamination on Chemistry Analytes 5

46 . Collection, Processing. and Storage Factors that Affect Chemistry Results . . . . . . . . . . . . . . . . . . . . . . . . . 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 . Factors that Affect Enzyme Reactions 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 . Differences in Analyte Concentrations 5

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 . Differences in Electrolyte Concentrations 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 . ChemistryPanels t

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 . ChemistryTests 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 . Enzymes 6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 . Summary of Diagnostic Enzymology i 54.CKandLDIsoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 . Cardiac Markers 7

xii CONTENTS

56 . Regulation of Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 57 . Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 58 . Tests for Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 . 'Ifrpical Laboratory Findings in Uncontrolled Diabetes Mellitus 79 60 . Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 61 . Characteristics of Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 62 . Lipid Levels (National Cholesterol Education Program Adult Treatment Panel) . . . . . . . . . . . . . . . . . . . . . 82 63 . Protein Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 64 . Common Serum Protein Electrophoresis Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 65 . Inborn Errors of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 66 . Bilirubin Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 67 . Bilirubin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 68 . Differential Diagnosis of Jaundice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 69 . Iron and Related Tests in Selected Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 70 . Structural Classes of Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 71 . Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 72.ThyroidTests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 73 . Thyroid Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Acid-Base Balance Terminology 99 75 . Acid-Base Imbalances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 76 . Arterial Blood Gases Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 77 . Blood Gas Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 78 . Blood Gas Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 79 . Sources of Error in Arterial Blood Gases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 80 . Therapeutic Drug Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 81 . Therapeutic Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.Toxicolo gy 108 83 . Visible Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.Spectrophotometry 111 85 . Chemistry Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

CONTENTS xiii

86 . Automated Chemistry Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 87 . Calculated Chemistry Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 88 . Chemistry Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

SECTION 3: CLINICAL MICROBIOLOGY REVIEW ............................................. 123 89 . Biosafety Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 90 . Biologic Safety Cabinets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 . Sterilization and Disinfection 126 92 . Bacterial Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 93 . Specimen Collection Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 . Specimen Preservation and Storage 130 95.FragileOrganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 96 . Examples of Criteria for Rejection of Specimens in Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97. TheGramStain 133 98 . Staining Properties of Gram-Positives and Gram-Negatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 99 .vpesofMedia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

100 . Routine Primary Media for Aerobes and Facultative Anaerobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 101 . Media for Culture of Anaerobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 102 . Special Bateriologic Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 103 . Aerotolerance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 104 . Organisms Requiring lncubation in lncreased CO, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 105 . Gram-Positive Cocci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 106 . Summary of Tests for Identification of Gram-Positive Cocci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 107 . Gram-Positive Bacilli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 108 . Neisseria and Related Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 109 . Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 110 . Biochemical Tests for Identification of Enterobacteriareae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 111 . Antigens of Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 112 . Commonly Isolated Enterobacteriareae (Usual Reactions) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 113 . Summary of Key Reactions for Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 114 . Appearance of Enterobactenaceae on Selected Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 . Trematodes 225 147 . Blood and Tissue Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 148.Plasmodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 149 . Blood and Tissue Helminths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 150 . Parasites of the Urogenital Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 151 . Stains Used in Mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 152 . Media Used in Mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 153.Dermatophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 154.DimorphicFungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 155.Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 156 . Contaminants/Opportunistic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 157 . Fungal Pathogens by Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 . Viral Structure 243 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 . Viral Replication 244

160 . Human DNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 . . . . . . . . . 161 . Human RNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 246

162 . Common Viral Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 163 . Specimens for Viral Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 164 . Viruses that May Be Cultured from Clinical Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 165 . Methods Used to Diagnose Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 166 . Tissue Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 167 . Comparison of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

SECTION 4: HEMATOLOGY REVIEW ...................................................... 255 168 . Adult-Reference Ranges: Comparison of Conventional and SI Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 169 . Pediatric Hematologic Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 170 . Bloodcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 . Hematopoiesis 259 172 . Changes During Cell Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 173 . Erythrocytic Developmental Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262 174 . Asynchronous Erythropoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

xvi CONTENTS

. 175.Hemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264 176 . Hemoglobin Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 177 . Hemoglobin Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 178 . Troubleshooting the Wright's-Stained Blood Smear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 179 . RBCMorphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 180.RBCInclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 181 . Summary of RBC Inclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 182 . Erythrocyte Indices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 183 . Hemoglobinopathy vs . Thalassemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 184.Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 185 . Differentiation of Microcytic Hypochromic Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 186 . Anemia Due to Blood Loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 187 . Highlights of Granulocytic Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 188 . Normal Leukocytes of the Peripheral Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 189 . Leukocyte Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 190 . Quantitative Abnormalities of Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289

. 191 . Acute vs Chronic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 192 . Acute Nonlymphocytic Leukemia (ANLL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 193 . Acute Lymphoblastic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 194 . Chronic Myeloproliferative Disorders (CMPD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 195 . Leukemoid Reaction vs . Chronic Myelogenous Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 196 . Chronic Lymphoproliferative Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 197 . Plasma Cell Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 198 . Hematology Special Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 . Erythrocyte Sedimentation Rate (ESR) 299 200 . Other Manual Hematology Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 . Sources of Error in Selected Manual Hematology Tests 303 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 . Changes in Blood at Room Temperature 305

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 . Interfering Factors on Most Hematology Analyzers 306 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 204 Hematology Calculations 308

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : 205 . Overview of Hernostasis 312

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235 . Labeled Immunoassay Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 236 . Comparison of Radioimmunoassay and Enzyme Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 237 . ImmunoassayLabels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359 238 . Principles of Labeled Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 239 . Labeled Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 240 . Nontreponemal Tests for Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 241 . 'I'reponemal Tests for Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 242 . Tests for the Diagnosis of Infectious Mononucleosis (IM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 243 . Weil-Felix Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 244 . Antinuclear Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 245.HepatitisTests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370 246 . Hepatitis Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247. HIVTesting 372 248 . Other Serological Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 249 . Interpretation of Serological Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376 250 . Serology Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

SECTION 6: IMMUNOHEMATOLOGY REVIEW ............................................... 379 251 . Primary vs . Secondary Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 252 . 1gGvs.IgM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 253 . Signs of Antigen-Antibody Reactions in Blood Bank Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382 254 . Factors that Affect Agglutination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 255 . Antigen-Antibody Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384 256 . Antihuman Globulin Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 257 . Antiglobulin Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258.AandBAntigens 388 259 . ABO Genotypes and Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 260 . Using Punnett Square to Predict ABO ?frpe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 261 . Frequency of ABO Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262. ABOSystem 392 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263.ABOTyping 393

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295 . Blood Donor Deferrals (AABB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 296.BloodCollection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 297 . Anticoagulants/Additives/Rejuvenating Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433 298 . CPDA-1 Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 299 . Donor Serological Testing Required by AABB and/or FDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436 300 . Autologous Transfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437 301 . BloodComponents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 . Opensystem 442 303 . Changes in Stored Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443 304 . Blood Bank Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444 305 . FDA Required Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446

SECTION 7: URINALYSIS AND BODY FLUIDS REVIEW ......................................... 447 306 . Changes in Urine at Room Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448 307 . UrineVolume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 308 . Urine Color and Clarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450 309 . Chemical Urinalysis by Reagent Strip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 310 . Confirmatory/Supplemental Urine Chemistry Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455 311 . Effect of High Levels of Ascorbic Acid on Urinalysis Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457 312 . Cells in the Urine Sediment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 313 . Crystals Found in Acid or Neutral Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460 314 . Crystals Found in Alkaline Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462 315 . Casts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463 316 . Miscellaneous Urine Sediment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 317 . Renal Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466 31 8 . Cerebrospinal Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467 319 . Meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469 320 . BodyFluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470 321 . Transudates and Exudates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471 322 . CellsinBodyFluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472 323.BodyFluidCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

CONTENTS xxi

353 . Laboratory Operating Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509 354 . Payroll Hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510 355 . Break-Even Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511 356 . Reimbursement for Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512 357 . Legal Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514 358 . Competency-Based Instruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517 359 . The ABCs of Writing Behavioral Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518 360 . Learning Styles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520 361 . What Students Remember . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521 362 . Domains of Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522 363 . Bloom's Cognitive Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523 364 . The Seven Principles of Good Teaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524 365 . Planning for Instruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366. TheLessonPlan 526 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 . Instructional Methods 527

368.Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528 369 . Norm-Referenced vs . Criterion-Referenced Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529 370 . m e s of Test Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530 371 . Testing at Different Cognitive Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 532 372 . Changing Educational Paradigms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 373 . What Students Say About Their Best Teachers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534 374 . What Students Say About Their Worst Teachers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536 375 . Legal Issues for Educational Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537 376 . Test-Taking Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538

CONTENTS xxiii

324.SynovialFluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 . Synovial Fluid Crystals 475

326 . SemenAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476 327 . Amniotic Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478

........................................ SECTION 8: MANAGEMENT AND EDUCATION REVIEW 479 328.ManagementSkills . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480 329 . The Foundations of Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481 330 . The Management Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482 331 . Laboratory Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 . McGregor's Theory X and Theory Y 484 333 . Management Styles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485 334 . Maslow's Hierarchy of Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486 335 . Principles of Total Quality Management/Continuous Quality Improvement . . . . . . . . . . . . . . . . . . . . . . . . 487 336 . Team Problem-Solving Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488 337 . Federal Legislation Governing Hiring Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 . Federal Legislation Governing Employee Compensation 490 339 . Interview Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 . Components of a Criteria-Based Job Description 493 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Components of an Employee Performance Appraisal System 494

342 . Performance Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495 343 . Evaluation Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 . Testing Personnel Competency Assessment 497 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 . Documents to be Included in Personnel File 498

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 . Requirements of OSHA's Bloodborne Pathogen Standard 499 347 . Requirements for a Chemical Hygiene Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 348 . Requirements of OSHA's Formaldehyde Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 . Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) 502 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 . Test Complexity under CLIA '88 503

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 . Personnel Required in High-Complexity Laboratories under CLIA '88 506 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 . Laboratory Finances 507

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r . 1 264 . ABO Discrepancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 265 . Non-Group Specific Transfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 6 1 266 . Rh Genotypes and Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 7 1 267 . Using Punnett Square to Predict Rh Vpe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 8 ; 268 . RhAntigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 269 . Frequency of Rh Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 270 . FrequencyofRhGenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 271 . Breaking the Rh Secret Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 272.RhTyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 273 . Positive Rh Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404 274.RhTypingSera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 0 S i 275 . Selection of Rh v p e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 0 7 1 276 . Frequency of Other Selected Blood Group Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 0 8 ; 277 . ISystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 278 . Antibody Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411 279 . Antibody Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413 280 . lnterpretation of Antibody Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 4 i 281 . Cold Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 5 ; 282 . Compatibility Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 416 283.Crossmatches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 7 ' i 284 . The Major Crossmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 285 . The lncompatible Crossmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419 286 . Emergency Transfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420 287 . NewbornTesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 288 . Conditions for Reissue of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 2 ~ 289 . Transfusion Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423 290 . Transfusion Reaction Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425 291 . Transfusion-Associated Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426 292 . Hemolytic Disease of the Newborn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427 293 . Rh Immune Globulin (RhIG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294 . Allogeneic Blood Donor Criteria (AABB) 429

xx CONTENTS

206 . Coagulation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 207 . Summary of Coagulation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 208 . Inherited Factor Deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316 209 . Acquired Factor Deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 210 . Coagulation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 211 . Effect of Factor Deficiencies on PT and APTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 212 . Substitution Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 . Laboratory Findings in Selected Coagulation Abnormalities 324 214 . Disseminated Intravascular Coagulation vs . Primary Fibrinolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 215 . Anticoagulant Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 216 . Thrombolytic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 217 . Sources of Error in Coagulation Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 . Causes for Rejection of Specimens for Coagulation Testing 331

...................................................... SECTION 5: IMMUNOLOGY REVIEW 333 219 . ImmunoIogyTerms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 220 . Branches of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 221 . Types of Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.AdaptiveImmunity 339 223 . Cells of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 224.LymphoidOrgans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 . Laboratory Identification of Lymphocytes 343 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 Common Lymphocyte Markers 344

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 . Immunoglobulin Structure 345 228 . Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 . Complement 348 230 . ImmunePhenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 . Hypersensitivity Reactions 350 232 . Agglutination Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351 233 . Precipitation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 . Other Serological Methods 355

xviii CONTENTS

RBC Morphology

Abnormality Description Significance

Anisocytosis Variation in size Seen in many anemias

Macrocytes RBCs greater than 9 p,m Vitamin B,, deficiency, folic acid deficiency, pernicious anemia, hemolytic anemia, cirrhosis, alcoholism. Nor- mal in newborns.

Microcytes RBCs less than 6 p,m Iron-deficiency anemia, thalassemia

Poikilocytosis Variation in shape Seen in many anemias

Elliptocytes/0valocytes Oval or pencil-shaped Hereditary ovalocytosis, various anemias - - - - -

Crenated RBCs Round cell with knobby, Osmotic imbalance. If seen in most cells in thin part of uniform projections smear, don't report. Probably artifact due to excess an-

ticoagulant or slow drying.

Burr cells (echinocytes) Round cell with spiny, Damage to RBC membrane, uremia, carcinoma of unevenly spaced projections. stomach, bleeding peptic/gastric ulcers Variable number in different fields.

Acanthocytes Shrunken cells with irregular, Damage to RBC membrane, alcoholic cirrhosis, post- spiny projections splenectomy, abetalipoproteinemia

Continued

270 HEMATOLOGY REVIEW

- -- -- - -

RBC Morphology Continued


Polychromasia Bluish-gray color Young RBCs. Reticulocytes (retics) with supravital stain. Sign of active erythropoiesis. 1-2 % in normal adult. Increased with acute blood loss, hemolytic anemia, following treatment for iron deficiency or pernicious anemia.

Target cells (codocytesl Bull's-eye, "Mexican hat cell" Hemoglobinopathies, thalassemia, alcoholism, postsplenectomy. May be artifact if observed in only one part of smear.

Stornatocytes RBC with slitlike central pallor Hereditary stomatocytosis, hereditary spherocytosis, thalassemia, alcoholic cirrhosis, Rh null disease. May be artifact in parts of smear that are too thin or too thick.

Spherocytes Small, dark-staining RBCs Damaged RBC membrane. Hereditary spherocytosis, without central pallor autoantibodies, burns, hemoglobinopathies, hemoly-

sis, ABO hemolytic disease of the newborn, incompatible blood transfusion, transfusion of stored blood. A few are normal due to aging of RBCs.

Rouleaux ~-~ - -

RBCs resemble stack of coins Serum protein abnormality; e.g., increased globulins or fibrinogen. Seen in multiple myeloma and macroglobulinemia. May be artifact due to delay in spreading drop of blood or smear that is too thick.

Agglutination RBCs in irregular clumps Autoantibodies, cold autoagglutinin


- -- --- -- - - --- - -- -- --

RBC Inclusions

inclusion Stain Descrintion Explanation Significance Conditions

Basophilic Wright's and Multiple, irregular stippling new methy- purple inclusions

lene blue evenly distributed throughout cell

Howell-Jolly Wright's and Round, purple, bodies new methy- 1-2 k m in dia-

lene blue meter. Usually only one per cell.

Aggregation of Coarse: exposure RNA (ribosomes) to lead. Fine:

young RBC.

Nuclear rem- Usually pitted nants (DNA) by spleen. Seen

with accelerated or abnormal erythropoiesis.

Exposure to lead, accelerated or abnormal hemoglobin synthesis, thalassemia

Postsplenectomy, thalassemia, hemolytic and megaloblastic anemias, sickle cell anemia

Cabot rings Wright's Reddish purple May be part of Rapid blood Pernicious anemia, rings or figure mitotic spindle, regeneration, thalassemia, postsplenec- eights remnant of abnormal tomy, lead poisoning

microtubules, or erythropoiesis fragment of nuclear membrane

Pappen- heimer bodies

Wright's Small purplish- Iron particles Faulty iron Sideroblastic anemias, post- (siderotic blue granules. Vary utilization splenectomy, thalassemia, granules in size, shape, sickle cell anemia, with Prussian number. Usually at hemochromatosis blue stain.) periphery in clusters.

Continued

HEMATOLOGY REVIEW 273

--

Summary of RBC Inclusions

Inclusion Wright's Stain New Methylene Blue Stain Prussian Blue Stain

Reticulum Cell appears polychromatophilic Yes No

Howell-Jolly bodies Yes Yes No

Pappenheimer bodies Yes Yes Yes

Siderotic granules Yes, but called Pappenheimer bodies Yes Yes - -

Heinz bodies No Yes No


Erythrocyte Indices

Index Definition Formula Reference Ranges Comments

Mean cor- Average volume HCT (%) Male = 80-94 fL Useful in classification of puscular of the RBC MCV = Female = 81-99 fL anemias. RBCs of normal volume RBCs (X 1012/L) MCV are described as (MCV) normocytic. RBCs with MCV

>lo0 appear macrocytic on the blood smear. RBCs with MCV <80 appear microcytic on the blood smear. MCV is an average. A combination of microcytes and macrocytes may be observed with a normal MCV.

Mean cor- Average weight Hgb (g/dL) X 10 27-31 pg Varies in proportion to MCV. puscular of hemoglobin MCH = Least useful of the indices. hemoglobin in the average RBC (X 1012/L) (MCH) RBC

Continued


-- -

- -

Hemoglobinopathy vs. Thalassemia

Hemoglobinopathy Thalassemia

Abnormality Qualitative abnormality. Abnormality in amino Quantitative abnormality. Amino acid acid sequence of globin chain, but not in amount sequence of globill chains is normal, but of globin produced. there is underproduction of one or more

globin chains.

Examples Sickle cell anemia and trait, hemoglobin C Beta thalassemia major and minor disease and trait


- - - -- - - -

-

Anemias

Hemoglobin Anemia Etiology Classification Blood Smear Electrophoresis Other

Iron deficiency Inadequate iron Microcytic, Aniso, poik, Normal for hemoglobin hypochromic hypochromic

, i synthesis^ microcytes

Sideroblastic ~nzymat ic Microcytic, Dimorphic Normal anemia defect in heme hypochromic RBCs, Pappen-

synthesis heimer bodies, basophilic stippling

.1 RBC, ? red-cell distribution width (RDW), & serum iron, '? total iron- binding capacity (TIBC) ,

serum ferritin

Siderocytes, ? serum iron, 4 TIBC, '? serum ferritin. retics. ? ringed sideroblasts in marrow.

- - - - -

Anemia of Defective iron Usually normo- Normal or Normal -1 serum iron, normal chronic utilization cytic, normo- hypochromic, TIBC, ? serum ferritin, diseases chromic. One- microcytic and free erythroc te

fourth to one- RBCs protoporphyrin. Yretics third may be microcytic, hypochromic

Beta thalas- Decreased pro- Microcytic, Aniso, poik, >90% A, ? RBC, normal RDW, semia minor duction of hypochromic hypochromic 3.5-7% A,, normal serum iron and

chains (hetero- microcytes, F may be slightly TIBC zygous) target cells, increased

Continued


-- -

Anemias Continued


Sickle cell Inheritance of anemia (SS) sickle cell gene

from both parents. Substitution of valine for glutamic acid in sixth position of B chain.

Normocytic, Aniso, poik, >80% S, normochromic sickle cells, frag- 1-20% F,

mented cells, 2-4.5 % A,, target cells, nu- no A cleated RBCs, spherocytes, Howell-Jolly bodies, basophilic stippling, siderotic granules, polychromasia.

Hgb S polymerizes under decreased 0, and decreased blood pH. Disease not evident in newborn because of ? Hgb F. Positive solubility test. Retics 5-20%. May have leukocytosis with shift to left and thrombocytosis.

Sickle cell trait (AS)

Inheritance of Normocytic, Occasional target 50-65 % A, Positive solubility test sickle cell gene normochromic cell. No sickle 35-45 % S, from one parent cells unless normal F,

hypoxic. normal to slightly increased A,

Continued


--- --

Anemias Continued


and polychromasia. Spherocytes.

? indirect bilirubin, ? osmotic fragility

Autoimmune Autoantibodies Normocytic, Polychromasia, Normal ? retics, ? indirect hemolytic normochromic spherocytes, bilirubin, anemia nucleated RBCs haptoglobin,

positive direct antiglobulin test


-

--

Differentiation of Microcytic Hypochromic Anemias

Free Percent Erythrocyte Retics

Serum satura- Serum Protopor- (Before Iron TZBC tion Ferritin phyrin (FEP) RDW lkeatment) Other

Iron 1 t deficiency

t -1 or Normal Normal hemoglobin

electrophoresis

Beta Normal Normal Normal Normal Normal Normal 1 or Increased Hgb A, thalassemia Normal minor

Anemia of 1 Normal Normal ? chronic disease

t Normal

Sideroblastic T 1 7 t Variable t 1 Dimorphic RBC anemia population,

siderotic granules, bas0 hilic stippling, S ringed sideroblasts in marrow


RBC Morphology Continued

A bnonnality Description Significance

Schistocytes RBC fragments, helmets, Fragmentation of RBCs. Seen with disseminated triangular cells intravascular coagulation (DIC), hemolysis, artificial

heart valves, burns, microangiopathic hemolytic anemia

- - ~

Blister cells RBCs with one or more Microangiopathic hemolytic anemia, pulmonary vacuoles emboli in sickle cell anemia

Sickle cells Crescent, S- or C-shaped, boat- (drepanocytes) shaped, oat-shaped Sickle cell anemia

Hemoglobin C crystals Blunt, six-sided, dark staining Hemoglobin C disease projection. "Bar of gold." "Washington monument."

Hemoglobin SC crystals Glovelike intracellular crystals Hemoglobin SC disease

Teardrops (dacryocytes) Teardrop-shaped Myelofibrosis, various anemias

Hypochromia Central pallor greater than Iron-deficiency anemia, thalassemia one-third cell diameter

Anisochromia Mixture of normochromic and Dimorphic anemia, post-transfusion hypochromic RBCs

Continued


Anemia Due to Blood Loss

Acute Blood Loss Chronic Blood Loss

RBCs Normocytic, normochromic Microcytic, hypochromic (due to iron deficiency)

WBCs Increased with shift to left for about 2-4 days Normal

Reticulocytes Increased within 3-5 days. Peak around 10 days. Normal - - - - - - - - - - - - - - - - - - - -

Hematocrit Steady during first few hours due to vaso- Decreased and constriction and other compensatory hemoglobin mechanisms. Full extent of hemorrhage evident

within 48-72 hours.

Other Platelets increase within 1 hour. Nucleated Decreased serum iron and ferritin RBCs may be released.


-

RBC Inclusions Continued

Inclusion Stain Description Explanation Significance Conditions

Siderotic Prussian Blue granules of Aggregates of Faulty iron utili- Sideroblastic anemias, post- granules blue varying size and iron particles zation in hemo- splenectorny, thalassemia,

shape globin sy~ithesis sickle cell anemia, he~riochromatosis

Reticulocytes New methy- Blue-staining Residual RNA lene blue network (ribosomes) (polychromasia on Wright's stain)

>2% increased Hemolytic anemia, blood erythropoiesis. loss, following treatment <0.1% decreased for iron deficiency or erythropoiesis. megaloblastic anemia

Heinz bodies Supravital Round blue Precipitated, stain; e.g., inclusions, varying oxidized, crystal violet, sizes, close to cell denatured brilliant membrane. May hemoglobin cresyl blue, be more than one. methylene blue


Normal during Glucose-6-phosphate aging but pitted dehydrogenase (G6PD) by spleen deficiencies, unstable

hemoglobins, chemical injury to RRCs, drug- induced hemolytic anemia

Highlights of Granulocytic Maturation

Stage Key Characteristics

Myeloblast 15-20 km. Small amount of dark blue cytoplasm. Usually no granules. Nucleus has delicate chromatin with nucleoli. QgSAKs J kc (= ~b,?

Promyelocyte Similar to myeloblast but has primary (nonspecific) granules

Myelocyte -

Presence of secondary (specific) granules (eosinophilic, basophilic, or neutrophilic). Last stage to divide.

Metamyelocyte Nucleus begins to indent.

Band Nuclear indentation is more than half.

Segmented Neutrophil ?tYo to five nuclear lobes connected by thin strands of chromatin.


- - -- - -

-- -

-

Erythrocyte Indices Continued

Index Definition Formula Reference Ranges Comments

Mean cor- Average Hgb (g/dL) X 100 32-36 g/dL RBCs with normal MCHC are puscular concentration of MCHC =

HCT (%) described as normochromic.

hemoglobin hemoglobin per They have an area of central concentration unit volume of pallor approximately one-third (MCHC) RBCs the diameter of the cell. The

MCHC is decreased in hypochromic cells and the area of central pallor is increased. RBCs cannot accommodate more than 37 g/dL of hemoglobin. Higher levels are in- dicative of problems with the specimen (hyperlipidemia, cold agglutinins) or the instrument.


- -- - -

Normal Leukocytes of the Peripheral Blood

Cell Size Nucleus

Adult Reference Adult Reference Range (Relative Range (Absolute

Cytoplasm Count) Count)

Neutrophil 10-15 pm Segmented. Two to five Pinkish-tan with 50-70% 2.5-7.0 x 109/L lobes connected by thread- neutrophilic like filament of chromatin. granules

Eosinophil 12-16pm Band-shaped or seg- Large red 1-3 % 0.05-0.30 X 1OY/L mented into two lobes granules

Basophil 10-14km Usually difficult to see Dark purple 0-1 % because of overlying granules granules

Monocyte 12-22 wm Round, horseshoe-shaped, Gray-blue with 2 4 % or lobulated. Convoluted. indistinct uink Loose strands of chromatin.

granules. Vacuoles. Occasional pseudopods.

Lymphocyte 6-8 pm or Round or oval. Dense Sparse to 20-40 % 1-4 x l o 9 / ~ 8-15pm blocks of chromatin. abundant. Sky

Indistinct chromatin/ blue. May contain parachromatin separation a few azurophilic

granules.


Anemias Continued


basophilic stippling

Beta thalas- Decreased pro- Microcytic, Marked aniso Little or no A, MCV 50-60 fL, retics semia major duction of p hypochromic and poik, hypo- up to 98 % F. 2-8 % . Hgb F distribu- (Cooley's chains (homo- chromic micro- tion is heterogeneous anemia) zygous) cytes, target among RBCs.

cells, ovalocytes, nucleated RBCs, basophilic stippling

Pernicious Reduced intrinsic Macrocytic Macro-ovalocytes, Normal Achlorhydria, factor secondary teardrops, hyper- .L serum B,,, to gastric atrophy. segmentation, .1 intrinsic factor, Deficiency of d, WBCs, abnormal Schilling test, vitamin B,, irn- .1 platelets t lactate dehydrogenase pairs DNA (LD) synthesis.

Continued


- - --

Leukocyte Abnormalities


Shift to the left Presence of immature granulocytes in peripheral blood Bacterial infection, inflammation --- - -- - - ~-~

Toxic granulation Dark staining granules in cytoplasm of neutrophils Infection, inflammation

Dohle bodies Light blue patches in cytoplasm of neutrophils Infection, burns

Vacuolization Phagocytic vacuoles in cytoplasm of neutrophils Septicemia, drugs, toxins, radiation

Hypersegmentation More than 5 % of segmented neutrophils with five- One of first signs of pernicious lobed nuclei or any with more than five lobes anemia

Pelger-Huet anomaly Most neutrophils have round or bilobed nuclei. Inherited disorder. No clinical effect. May be misinterpreted as a shift to the left.

Auer rods Red needles in cytoplasm of leukemic myeloblasts and Nonlymphocytic leukemia monoblasts

- - - - - - - --- - - - - - - - -

Variant lymphocytes One or more of the following: large size, elongated or Viral infections (e.g., infectious (atypical or reactive) indented nucleus, immature chromatin, increased mononucleosis, cytomegalovirus)

parachromatin, nucleoli, increased cytoplasm, dark blue or very pale cytoplasm, peripheral basophilia, scalloped edges due to cytoplasm being indented by adjacent RBCs, frothy appearance, many azurophilic granules


- -- - ---

Anemias Continued


Hemoglobin Inheritance of C disease gene for Hgb C (CC) from both

parents. Lysine is substituted for glutamic acid in the sixth position of p chain.

Normocytic, Aniso, poik, >90 C, normochromic slight hypochro- <7% F, no A

masia, many target cells, fragmented cells, folded cells ("pocketbook cells "), occasional Hgb C crystals, occasional micro- spherocytes

Hemoglobin Inheritance of Normocytic, Many target cells 50-60% A, C trait (AC) gene for Hgb C normochromic 30-40% C

from one parent.

SC disease Inheritance of Normocytic, Target cells, Equal amounts Positive solubility test (sc) one sickle cell normochromic pocketbook cells, of C and S,

gene and one occasional SC normal to 7 % F, Hgb C gene crystals ("gloves") no A.

Hereditary Defect of cell Normocytic, Varying degrees Normal T MCHC, ? retics, spherocytosis membrane normochromic of aniso, poik, k haptoglobin,

Continued


- -- -- - - - - - -- - - --

Quantitative Abnormalities of Leukocytes

Neutrophilia Neutropenia Lymphocytosis Monocytosis Eosinophilia Basophilia

Bacterial Acute infection Infectious Convalescence from Allergies Chronic myelo- infection Antibodies mononucleosis viral infections Skin diseases genous leukemia

Inflammation Drugs Cytomegalovirus Chronic infections Parasitic Polycythemia vera Hemorrhage Chemicals Whooping cough TB infections Hemolysis Radiation Acute infectious Subacute bacterial Stress lymphocytosis endocarditis

Parasitic infections Rickettsia1 infections


Acute vs. Chronic Leukemia

Acute Chronic

Age Mostly children and young adults Mostly middle-aged and elderly

Onset Sudden Insidious

Median survival 3 months time if untreated

- ~

2-6 years

White count Usually increased, but may be normal Increased or decreased

Platelets Low to markedly decreased Normal or increased at first. Decreased in late stages.

Leukocytes Many blasts More mature cells

Anemia Normocytic, normochromic None until late in disease

lleatment Chemotherapy, bone marrow transplant Chemotherapy or none

Cause of death Infection, hemorrhage Infection, hemorrhage

Diagnosis Peripheral blood smear, bone marrow Peripheral blood smear examination, cytochemical stains, surface markers, chromosome analysis, electron microscopy

Other Majority go into blast crisis


- - - ---

Acute Nonlymphocytic Leukemia (ANLL)

I)rpe Name Principle Featurels)

AML (MO) Acute myelogenous Undifferentiated blasts without maturation

AML (Ml) Acute myelogenous with Predominance of myeloblasts with minimal maturation. Some promyelo- minimal maturation cytes may be present. Auer rods may be present. Pseudo Pelger-Huet

anomaly may be present.

AML (M2) Acute myelogenous Myeloblasts predominate. Some maturation to or beyond promyelocyte with maturation stage. Auer rods are common. Most comrnon type of acute myelogenous

leukemia.

APL (M3) Acute promyelocytic The promyelocyte is the predominant cell. Myeloblasts and myelocytes may be present. Auer rods are common. High incidence of DlC. Uncom- mon type.

AMML (M4) Acute myelomonocytic Early myelogenous cells predominate with approximately 20% monocytic cells. Blast may have indented and convoluted nuclei. Auer rods may be present. Pseudo Pelger-Huet anomaly may be present. Second most corn- mon type.

AMoL (M5) Acute monocytic M5a has a predominance of monoblasts. M5b has promonocytes and monocytes as well. Uncommon types\ p I+; rc ; ; 4-c &Sbase

I

EL (M6) Acute erythroleukemia Erythroblasts with megaloblastoid changes and other d z a s t i c features, (Di Guglielmo syndrome) myeloblasts, and promyelocytes. Auer rods may be present.

MegL (M7) Acute megakaryoblastic Proliferation of megakaryoblasts and atypical megakaryocytes. Megakary- ocytic fragments may be seen in the peripheral blood. Uncommon type.

HEMATOLOGY KEVIEW 291

- . - - - -- - - - - - - - - - - - - - - - -

Acute Lymphoblastic Leukemia

French-American-British (FAB) Morphologic Classification

L1 (Small cell homogeneous) Uniform population of small lymphoblasts with scanty cytoplasm, round nuclei with occasional clefting, homogeneous chromatin, inconspicuous nucleoli. Children.

~ - -

L2 (Large cell heterogeneous) Heterogeneous population of large, pleomorphic lymphoblasts with nuclear clefting and indentation. Older children and adults.

'\(~urkitt type) Uniform population of relatively large lymphoblasts with deeply basophilic cytoplasm, vacuoles, and round to oval nuclei without indentations. Sec-

6 ' J ~ ~ i n r s L \ jm~\*ne ondary to Burkitt's lymphoma.

Immunologic Marker Classification

B-cell ALL 1 5 % of ALL. Corresponds to FAB type L3.

Pre-B cell ALL 10-15% of ALL. Most are FAB type L1. -- - - - - - pp - - - - - - - -

Pro-B cell ALL 85% of childhood ALL and 75 % of adult ALL. FAB type L1 or LZ. Highest remission rate.

T-cell ALL 15% of ALL. FAB type L1 or L2. Poor prognosis.


Chronic Myeloproliferative Disorders (CMPD)

Disorder Other Names Principle Feature - - - - - - - - -

Chronic myelogenous Chronic granulocytic Marked leukocytosis with an increase in mature and leukemia (CML) leukemia immature cells of the granulocytic series. Thrombocytosis is

common. Philadelphia chromosome may be found in malignant cells. Anemia.

Agnogenic rnyeloid Idiopathic myelofibrosis. Bone marrow fibrosis. Extramedullary hematopoiesis. All metaplasia (AMM) Myelofibrosis with myeloid stages of myeloid maturation in peripheral blood, including

metaplasia. immature eosinophils and basophils. Nucleated RBCs. Teardrop RBCs. Defective platelets.

Polycythemia Vera (PV)

Primary polycythemia. Increase in all cellular bone marrow elements. Increased Erythremia. hemoglobin and hernatocrit. Increased leukocyte alkaline

phosphatase (LAP).

Essential thrombo- Platelets >lo00 X 109/L. Marked platelet anisocytosis. cythernia (ET) Occasional megakaryocyte fragments.


- -

Leukemoid Reaction vs. Chronic Myelogenous Leukemia

Lenkemoid Reaction CML

Peripheral blood smear Shift to the left (blasts rare), toxic granulation, Shift to the left with blasts, Dohle bodies eosinophilia, basophilia

LAP score High Low

Philadelphia chromosome Negative Usually positive


- -- -- -- .

-

Chronic Lymphoproliferative Disorders

Leukemia Key Characteristics

Chronic lymphocytic Most common leukemia in adults. B-CLL is most common. Lymphocytosis with relatively leukemia (CLL) mature lymphocytes. Smudge cells. May be accompanied by anemia, thrombocytopenia,

and production of autoantibodies (autoimmune hemolytic anemia or idiopathic thrombocytopenia purpura).

Hairy cell leukemia Hairy cells: scant to abundant, agranular, light grayish-blue cytoplasm with irregular hair- like projections. Round to oval nuclei with loose chromatin. Pancytopenia.


- -

- - -- - -

Plasma Cell Disorders

Disorder Key Characteristics

Multiple myeloma Malignant plasma cells in marrow. Rare abnormal plasma cell on peripheral smear. Normo- cytic, normochromic anemia. Rouleaux on blood smear. Elevated erythrocyte sedimentation rate due to increased globulins. M spike on serum protein electrophoresis (monoclonal gammopathy). May have Bence Jones proteinuria. Lytic bone disease.

- - - - -- - -

Plasma cell leukemia Abnormal plasma cells in peripheral blood (> 2 X lo9/ L). Pancytopenia. Rouleaux. Mono- clonal gammopathy.

Waldenstrom's Monoclonal gammopathy due to increased IgM. Rare plasmacytoid lymphocytes or plasma macroglobulinemia cells on peripheral smear. Rouleaux. May have Bence Jones proteinuria and cryoglobulins.


-- - --

Hematology Special Stains

Stain Detects Significance -

New methylene blue RNA Used for reticulocyte counts

Prussian blue Iron Detects siderocytes

Peroxidase (myelo- Peroxidase found in primary Differentiates AML M1, M2, and M3 (positive) peroxidase) granules and Auer rods from ALL (negative)

Sudan black Lipids in primary and secondary Differentiates AML M1, M2, and M3 (positive) granules of granulocytes from ALL (negative)

Napthol ASD chloro- Esterase i n primary granules Differentiates AML M1, M2, and M3 (positive) acetate esterase of myeloid cells from ALL (negative)

a-naphthyl acetate Nonspecific esterase in monocyte Strong positive in rnonocytic cells. Inhibited by esterase or a-naphthyl precursors addition of sodium fluoride. Positive in M4 and butyrate esterase MS. Negative in ALL.

Combined esterase Esterase enzymes in myeloid and Used to evaluate the ratio of myelocytic and monocy tic cells nlonocytic cells on a smear based on different

color of staining. Differentiates M4 and MS.

TdT Primitive lymphoid cells Activity is elevated in L1 and L2.

Periodic acid-Schiff (PAS) Glycogen RBC precursors in erythroleukernia stain in- tensely. Normal RRCs and normal RBC precursors are negative. Large coarse clumps in lymphoblasts in ALL.

Continued


Hematology Special Stains Continued

Stain Detects Significance

Tartrate resistant acid Tartrate resistant acid phosphatase Hairy cell leukemia phosphatase (TRAP)

Leukocyte alkaline Alkaline phosphatase in bands and Increased in leukemoid reaction and poly- phosphatase (LAP) segmented neutrophils cythemia vera. Decreased in CML. Score 100

segmented neutrophils and bands from 0-4 + and total. Normal: 20-100.


--

Erythrocyte Sedimentation Rate (ESR)

Measures Amount of settling of RBCs in a column of anticoagulated whole blood

Clinical significance Nonspecific indicator of inflammation. Increased in acute and chronic infections, rheumatic fever, rheumatoid arthritis, myocardial infarction, TB, subacute bacterial endocarditis

Methods Wintrobe and Westergren. Westergren is preferred. Longer column increases sensitivity.

Sources of error

Excess anticoagulant RBCs shrink. ESR J.

Hemolysis RBC column is decreased. ESR J.

Specimen at room temperature RBCs swell. ESR J. more than 4 hours

Room temperature below 22"-27°C Cells settle more slowly. ESR -1.

Room temperature above 22"-27°C Cells settle faster. ESR T.

Tilted tube ESR T. A 3" angle from vertical can accelerate ESR by 30%.

Inclusion of buffy coat in reading ESR 4

Inaccurate reading of scale ESR could be increased or decreased.

Bubbles in column ESR 4 Continued


- -- -- - - - p-p

Erythrocyte Sedimentation Rate (ESR) Continued

Inaccurate timing ESR could be increased or decreased.

Anemia Change in RBC:plasma ratio favors rouleaux formation. ESR 1'. Polycy themia Increased viscosity. ESR J.

Anisocytosis Cells don't rouleaux as readily. ESR J.

Poi kilocy tosis Cells don't rouleaux as readily. ESR J.

Agglutinated RBCs Agglutinates settle faster. ESR '?. Macrocytosis Large cells settle faster. ESR ?.

Microcytosis Small cells settle more slowly. ESR J.

Increased fibrinogen or globulins Favors rouleaux formation. ESR ?.


- - - - -

- --

Other Manual Hematology Procedures

Manual white blood cell Acetic acid is used as diluent to lyse RBCs. WBCs are counted in a Neubauer (WBC) count hemacytometer. Nucleated RBCs can't be distinguished from WBCs.

Eosinophil count Phloxine stains eosinophils red. Cells are counted in a special hemacytometer with a larger volume than a Neubauer chamber (Fuchs-Rosenthal or Speirs-Levy).

Hematocrit Packed cell volume (PCV). Microhematocrit tubes are centrifuged at 10,000 rpm for 5 minutes. The ratio of the volume occupied by the RBCs to the volume of whole blood is determined. Values may be slightly higher than automated hematocrit (HCT) due to trapped plasma. (Automated HCT is a calculated value).

Hemoglobin Cyanmethemoglobin method. Potassium cyanide and potassium ferricyanide reagent (Drabkin's). Potassium ferricyanide oxidizes ferrous ions to ferric ions, forming methemoglobin. Methemoglobin reacts with cyanide ions to form cyanmethemoglobin. Read at 540 nm. A commercially prepared standard is used to prepare a standard curve. Measures all forms of hemoglobin except sulfhemoglobin.

Sugar water test (sucrose Screening test for paroxysmal nocturnal hemoglobinuria (PNH). Sucrose provides a hemolysis) medium of low ionic strength that promotes the binding of complement. PNH RBCs

are complement-sensitive and lyse. Normal RBCs are unaffected.

Ham test (acid-serum test) For confirmation of PNH. Serum is acidified, which activates complement. PNH RBCs lyse. Normal RBCs are unaffected.

Hemoglobin A, by Screening test for beta thalassemia minor (? Hgb A,) microcolumn

Continued

HEMATOLOGY REVlEW 301

Other Manual Hematology Procedures Continued

Hemoglobin F Alkali denaturation: Hgb A is denatured; Hgb F is not. Acid elution: Hgb A is eluted and RBCs appear as ghost cells. Hgb F resists acid and KBCs with a high concentration stain pink. Hgb F is increased in hereditary persistence of fetal hemoglobin (HPFH), sickle cell anemia, thalassemia. In thalassemia there is a heterocellular distribution of Hgb F (some cells contain Hgb F and some don't). HPFH can be pan- cellular (uniform distribution of Hgb F among the RBCs) or heterocellular.

Solubility test for sickling Sickling hemoglobins produce a turbid solution when mixed with sodium dithion- hemoglobin ite. Screening test only. Not specific for Hgb S. Does not differentiate SS from AS.

Confirm with hemoglobin electrophoresis.

Osmotic fragility Measures the ability of RRCs to take up water without lysing. RBCs are incubated with various concentrations of NaC1. The amount of hemolysis is determined by reading the absorbance of the supernatant from each tube. Normal = hemolysis beginning at 0.45% and complete at 0.30%. Increased in hereditary spherocytosis. Decreased with target cells, sickle cell anemia, iron deficiency anemia, thalassemia.

LE prep Outdated test for diagnosis of lupus erythematosus. Antinuclear antibody in patient's serum digests nucleus of damaged WBC, which is then phagocytized by a neutrophil or monocyte.

Malaria smears Examine thin and thick smears. Thick smear concentrates parasites and is used to screen. It is not fixed, so RBCs lyse, making detection easier. Thin smear must be examined to speciate. Refer to Section 3 for speciation.


- - - - - -- - -

Sources of Error in Selected Manual Hematology Tests

Manual Hemoglobin

Lipemia Value is increased because of turbidity (higher absorbance). Use patient plasma in reagent to blank.

WBC >30 X 109/L Value is increased because of turbidity (higher absorbance). Centrifuge reaction tube and read absorbance of supernatant.

Hgb S or C Cells are resistant to lysis. Value is increased because of turbidity (higher absorbance). Dilute mixture 1:2 with distilled water. Reread. Multiply result X 2.

Manual Hematocrit

Tourniquet left on too long Increased due to hemoconcentration

Excess anticoagulant Decreased due to shrinkage of RBCs

Hemolysis Decreased due to destruction of RBCs

Insufficient mixing Could be higher or lower, depending on portion sampled

Inadequate time or speed Decreased values due to insufficient packing of cells of centrifugation

Inclusion of buffy coat Increases value in reading

Parallax error Failure to look straight down when reading result. Could increase or decrease value.

Continued


Sources of Error in Selected Manual Hematology Tests Continued

Incorrect use of Could be higher or lower, depending on error hematocrit reader

- - - - - - -

Poikilocytosis Increased, due to trapped plasma

Manual Reticulocyte

Refractive artifacts Increased values. (RNA disappears when out of focus.)

Other inclusions Pappenheimer bodies, Howell-Jolly bodies, Heinz bodies are seen with supravital stain. Differentiate by other stains.

'hbe Solubility Test for Hgb S

Lipemia False-positive due to turbidity

Leukocytosis False-positive due to turbidity

Polycy themia False-positive

Increased globulins False-positive due to turbidity

Blood from infant less False-negative due to predominance of Hgb F than 6 months old

Recent transfusion False-negative

Hemoglobin less False-negative than 7 g/dL


Changes in Blood at Room Temperature

MCV t (due to RBCs swelling)

Hematocrit ? (due to t MCV)

MCHC L (due to t hematocrit)

ESR L (swollen RBCs don't rouleaux)

Osmotic fragility t WBC count L WBC morphology Necrobiotic cells, karyorrhexis (nuclear disintegration), degranulation, vacuolization


- -- - - -- -

Interfering Factors on Most Hematology Analyzers - ~

Condition Effect - -

Parameters Affected Resolution

WBC over Turbidity may Increases RBC. May Dilute blood and rerun to get accurate 50,000 increase Hgb. increase Hgb. WBC. Subtract WBC from RBC. Follow manufacturer's

WBCs are counted HCT and indices instructions for obtaining accurate Hgb. Perform spun and sized as RBCs. inaccurate. HCT. Recalculate indices.

Cold Agglutinates are Decreases RBC. Prewarm blood to 37°C. and rerun. agglutinins counted and sized Increases MCV,

as RBCs. MCH, MCHC. HCT inaccurate.

Nucleated Some analyzers Increases WBC. Uncorrected WBC X 100 RBCs count as WBCs. Corrected WBC =

100 + NRBCs per 100 WBCs

Lipemia lbrbidity may affect May increase Hgb, Follow manufacturer's instructions for obtaining Hgb reading. MCH, MCHC. accurate Hgb. Recalculate indices.

Hemolysis RBCs are lysed. Decreases RBC and If hemolysis is in vitro, recollect. HCT. Increases MCH and MCHC.

Giant plate- Platelet clumps are Decreases platelets Examine blood film. For platelet clumps or satellitism, lets, plate- counted as WBCs. (PLT). recollect in sodium citrate. Multiply by 1.1 dilution fac- let clumps, Increases WBC. tor. Manual counts or film estimates may be required. or satellitism


-- - - -- - - - - - - -

Continued

-

Interfering Factors on Most Hematology Analyzers Continued

Condition Effect Parameters Affected Resolution

Old RBCs swell. Increases MCV. Recollect. specimen Platelets and Decreases PLT. Automated

WBCs degenerate. differential count (auto diff) may be inaccurate.

- - -

Schistocytes, Not counted as RBCs. Decreases RBC. HCT and Manual counts. Spun HCT. microcytes May be counted a s PLT. indices may be inaccurate. Recalculate indices.

Resistant RBCs don't lyse. Increases WBC. Manual Hgb, adding water to lyse RBCs. RBCs (e.g., Counted as .WBCs. Hgb inaccurate. Manual WBC. Recalculate indices. Hgb S or C)

Fragile WBCs WBCs are damaged in Decreases WBC. May Manual counts. (e.g., chemo- analyzer. May be increase PLT. therapy) counted as PLT.


-

-- -

Hematology Calculations

Formula Example Calculation

Reticulocyte retics Der 1000 RBCs What is the reticulocyte count if reticulum is observed in

15 of 1000 RBC?

15 Retic % = - = 1.5 10

Reticuloc~te % retics in square A x 100 using Miller Disc = RBCs in square

What is the reticulocyte count if 60 reticulocytes are counted in square A and 300 RBCs are counted in square B?

60 X 100 Retic % = 300 = 2.2

Absolute What is the absolute reticulocyte count if the reticulocyte Reticulocyte count is 2% and the RBC is 5.2 X 1012/L? Count (ARC) retic % x RBC count (1012/L) X 1000 2 X 5.2 X 1000 ( x 109/~) =

100 ARC =

100 = 104 x 1 0 9 / ~

Corrected Reticulocyte Count (CRC) = retic % X

HCT(%) 4s

What is the corrected reticulocyte count if the uncorrected reticulocyte count is 5 % and the hematocrit is 36 % ?

36 C R C = 5 X - = 4 % 45

Continued


-

Hematology Calculations Continued


Reticulocvte What is the RPI if the corrected retic is 5% and the HCT ~roductidn corrected retic count is 35%; (Maturation time correction factor for HCT of Index (RPI) = 35% is 1.5).

maturation time correction factor* I 5

RPI = 1.5 = 3.3%

HCT (%) x 10 Calculate the MCV if the RBC is 3.0 X 10I2/L, the Hgb is MCV = 6 gm/dL, and the HCT is 20%.

RBC (1012/L) 20 X 10

MCV = ------- 3 = 66.7 fL

Hgb (g/dL) X 10 Calculate the MCH if the RBC is 3.0 X 1012/L, the Hgb is

MCH = RBC (1012/L) 6 gm/dL, and the HCT is 20 % .

6 X 10 MCH = -

3 = 20 pg.

Hgb (g/dL) X 100 Calculate the MCHC if the RBC is 3.0 X 1012/L, the Hgb is MCHC = HCT (%) 6 g~n/dL, and the HCT is 20 % .

6 X 100 - 30% MCHC = -

- *The maturation time correction factor is based on the patient's hematocrit and is obtained from a maturation timetable.

Continued




Rules of Three:

RBC X 3 = Hgb Z 0.5

Hgb X 3 = H C T ? 3 %

What should the hemoglobin be if the RBC is 4.1 x 1012/L?

Hgb = 4.1 X 3 = 12.3 % 0.5 = 11.8-12.8 g/dL

What should the hematocrit be if the hemoglobin is 12.3 g/dL?

HCT = 12.3 X 3 = 36.9 % 3 = 33.9-39.9%

Cells per mm3 (or pL) = No. of cells counted X Calculate the WBC count if blood is drawn to the 0.5 mark depth factor (always 10) x reciprocal of and diluent to the 11 mark in a WBC diluting pipet and dilution X reciprocal of area counted (mm2) 100 WBCs are counted in the 4 "W" squares of a Neubauer

hemacytometer.

Cells per pL = 100 X 10 x 20 X '/4 = 5000

(In a WBC diluting pipet, 0.5 to 11 is a dilution of 1:20 because the diluent up to the 1 mark on the stem does not mix with the blood.)

(Area counted is 4 mm2.)

Calculate the RBC count if 10 pL of whole blood are diluted in a Unopette containing 1.99 mL of diluent and 250 RBCs are counted in four corner and one center "R" squares of the middle square millimeter (subdivided into 25 squares) of a Neubauer hemacytometer.

Continued


Formula Examvle Calculation ~~~~~~ ~ p ~ ~ -

Cells per pL = 250 X 10 X 200 X 5 = 2,500,000

(Dilution is 0.01:2 or 1:200.)

(Area counted is 1/5 mmz.)

Absolute WBC = Total WBC x relative count Calculate the absolute lymphocyte count if the total WBC is (% from differential) 10 x 10'/L and there are 70% lymphocytes.

Absolute count = 10 X 0.70 = 7 X 109/L

The automated hematology analyzer reports a WBC of 30 X

Corrected uncorrected WBC x 100 lO9/L. The technologist counts 115 nucleated RBCs per 100 WBCs while performing the differential. What is the corrected

WBC = 100 + nucleated RBCs per 100 WBCs WBC. 30 X 100

Corrected WBC = + 115 = 14.0 X 109/L


- - - -

-

Overview of Hemostasis - - - - - - -

Primary hemostasis Vasoconstriction. Platelet adhesion to exposed subendothelial connective tissue. Platelet aggregation to form initial plug at injury site.

Secondary hemostasis Coagulation factors interact on platelet surface to produce fibrin. Fibrin stabilization by factor XIII.

Fibrinolysis Release of tissue plasminoge~l activator. Conversion of plasminogen to plasmin. Conversion of fibrin to fibrin degradation products (fibrin split products).


-

Coagulation Factors Continued

- ---- -

Present in Vit K Present in Adsorbed Present in

Name Required? Plasma? Plasma? Serum? Pathway Other

IX Christmas Yes Yes No Yes Intrinsic Deficiency = factor hemophilia B

X Stuart-Prower Yes Yes NO Yes Common factor

XI Plasma throm- No Yes Yes Yes Intrinsic Deficiency = boplastin hemophilia C antecedent

XI1 Hageman factor No Yes Yes Yes Intrinsic Glass activation factor. Not required in vivo. No clinical signs of deficiency.

XI11 Fibrin stabilizing factor

Stabilizes fibrin clot. Deficiency leads to poor wound heal- ing. Assay by urea solubility test.


Coagulation Factors

Present in Vit K Present in Adsorbed Present in

Name Required? Plasma? Plasma? Serum? Pathway Other

\ Fibrinogen No Yes Yes No Common Converted to fibrin by thrombin. Normal = 200-400 mg/dL.

I\ Prothrombin Yes Yes No No Common Precursor of thrombin

I11 Tissue throm- High molecular weight boplastin Extrinsic lipoprotein present in all

tissues. Liberated by trauma.

IV Ca2+ Intrinsic, Round by sodium citrate. extrinsic, Must be replaced by and common reagents.

V LabiIe factor No Yes Yes No Common Deteriorates at room temperature

VII Stable factor Yes Yes No Yes Extrinsic

VIII Antihemo- No Yes Yes No Intrinsic Deficiency = hemophilia philic factor A. Most common inher-

ited bleeding abnormality. Labile.

Continued

HEMATOLOGYREVIEW 313

- -

Summary of Coagulation Factors

Produced in liver All. (Von Willebrand's portion of VIII also produced in endothelial cells and megakaryocytes.)

Require vitamin K for 11, VII, IX, X (prothrombin group) synthesis

Affected by Cournadin 11, VII, IX, X (prothrombin group)

Consumed during clotting I, 11, V, VIII, XI11

Labile factors V, VIII

Contact group Prekallikrein [Fletcher factor), high-molecular-weight kininogen (Fitzgerald factor), XII, XI

Factors in extrinsic pathway 111, VII

Factors in intrinsic pathway Prekallikrein (Fletcher factor), high-molecular-weight kininogen (Fitzgerald factor), XII, XI, IX, VIII

Factors in common pathway X, V, 11, I


- - - - - - - - - -

- -

Inherited Factor Deficiencies

Factor Name of Disease Pearment Comments

I Afibrinogenemia, hypo- Cryoprecipitate Rare fibrinogenemia, dys- fibrinogenemia

11 Prothrombin deficiency Fresh frozen plasma Rare

V Labile factor deficiency Fresh frozen plasma (Owen's disease)

Autosomal recessive

VI I Factor VII deficiency Plasma or prothrombin complex Rare

VIII Hemophilia A Cryoprecipitate, factor VIII One of most common inherited coagula- concentrate, l-desamino-8- tion disorders. Sex-linked recessive.

D-arginine vasopressin (DDAVP) Occurs primarily i11 males. Mothers are carriers.

Von Willebrand's disease Cryoprecipitate, DDAVP One of most common inherited coagulation disorders. Deficiency of VW factor @art of factor VIII complex). Autosomal dominant. Both sexes are affected.

IX Hemophilia B Plasma or commercial Sex-linked recessive (Christmas disease) concentrates

Continued

316 HEMATOLOGY KEVIEW

Inherited Factor Deficiencies Continued

Factor Name of Disease meahnent Comments

X Stuart-Prower factor Fresh frozen plasma or Rare deficiency prothrombin complex

XI Hemophilia C Fresh frozen plasma Rare

XI1 Hageman trait None No bleeding disorders

XI11 Fibrin-stabilizing Plasma or lyophilized Rare factor deficiency placental factor XI11

HEMATOLOGYREVIEW 317

Acquired Factor Deficiencies

Condition Factors Affected

Liver disease Mild: 11, VII, IX, X Moderate: also V, VIII Severe: also I

Vitamin K deficiency 11, VII, IX, X

Coumadin therapy 11, VII, IX, X -

Disseminated intravascular coagulation I, 11, V, VIII, and platelets

Primary fibrinolysis I, V, VIII


- -

Coagulation Tests

Test Significance

Bleeding time Prolonged with platelet abnormalities, vascular disease. Prolonged by aspirin.

Clot retraction Decreased with thrombocytopenia and qualitative platelet defects. Infrequently performed.

Platelet aggregation Test of platelet adhesion, aggregation, and secretion. Detects qualitative platelet defects.

Prothrombin time (PT) Detects deficiencies in extrinsic and common pathways. Used to monitor Coumadin therapy. Report in INR (International Normalized Ratio).

Activated partial throm- Detects deficiencies in intrinsic and common pathways. Most common test to monitor boplastin time (APTT) heparin therapy. - - - - -

Activated coagulation Used for bedside monitoring of heparin therapy time (ACT)

Thrombin clotting Little used test to monitor heparin therapy time (TCT)

Fibrinogen Estimation of fibrinogen level

Urea solubility Factor XI11 screening test

D-Dimer Positive in DIC, deep vein thrombosis, pulmonary embolism, and after lytic therapy. Negative in primary fibrinolysis.

-

Continued

HEMATOLOGY REVIEW 3 19

-- - - --- - - - - - -

Coagulation Tests Continued

Test Significance

Fibrin(ogen) degradation Present in DIC, primary fibrinolysis, deep vein thrombosis, pulmonary embolism, after (split) products lytic therapy (FDP, FSP)

Plasminogen Precursor of plasmin. Decreased following lytic therapy, DIC, primary fibrinolysis.

Antithrombin 111 (AT-111) Heparin cofactor. Deficiencies associated with thrombosis.

Protein C and protein S Inhibitors of coagulation. Deficiencies associated with thromboembolic disorders.


- - p- - - - - - - - - - -- - -- -- - - -

Effect of Factor Deficiencies on PT and APTT

Factor Deficiency PT APTT

I Prolonged Prolonged

11 Prolonged Prolonged

V Prolonged Prolonged

VII Prolonged Normal

VIII Normal Prolonged

IX Normal Prolonged

X Prolonged Prolonged

XI Normal Prolonged

XI1 Normal Prolonged


- - - - - -

- -

Substitution Studies

PT APTT Deficiency Corrected By Not Corrected By

Abnormal Normal VII Fresh normal plasma Adsorbed plasma Serum

Normal Abnormal VIII

IX

XI

Fresh normal plasma Adsorbed plasma

Fresh normal plasma Serum

Fresh normal plasma Adsorbed plasma Serum


Serum

Adsorbed plasma


--

Continued

- --

Substitution Studies Continued

PT APTT --

Deficiency Corrected By ~- ~

Not Corrected By

Abnormal Abnormal I Fresh normal plasma Adsorbed plasma Serum



X Fresh normal plasma Adsorbed plasma Serum

To identify a factor deficiency, patient's plasma is mixed with plasma deficient in a single clotting factor. Failure of the factor- deficient plasma to correct the clotting time of the patient's plasma indicates that the patient's plasma is missing the same factor as the factor-deficient plasma.


-. -- - - -- -

Laboratory Findings in Selected Coagulation Abnormalities

--

Disease Laboratory Findings

Hemophilia A (classic hemophilia) Decreased factor VIII. APTT prolonged. PT normal. Bleeding time normal. Platelet count normal.

Hemophilia B APTT prolonged. PT normal. Bleeding time normal. Platelet count normal.

Von Willebrand's disease Bleeding time prolonged. Platelet aggregation abnormal with ristocetin. Factor VIII may be decreased. APTT may be prolonged. PT normal. Platelet count normal.

Glanzmann's thrombasthenia Clot retraction abnormal. Bleeding time prolonged. Platelet count normal. Platelet aggregation abnormal with ADP,

collagen, thrombin, epinephrine.

Continued


i -- -- - -- - -- - -

Laboratory Findings in Selected Coagulation Abnormalities Continued

Disease Laboratory Findings

Bernard-Soulier sy itdrome Bleeding time prolonged. Giant platelets. Platelet count decreased. Clot retraction normal.

Disseminated Intravascular Coagulation vs. Primary Fibrinolysis

DZC Primary Fibrinolysis

PT Prolonged Prolonged

APTT Prolonged Prolonged

Fibrinogen Decreased Decreased

Platelets Decreased Normal

FSP Present Present

D-Dimer Positive Negative

AT-111 Decreased Normal

RBC morphology Schistocytes Normal


-- - - -- - - - -

- - - - -

Anticoagulant Therapy

Heuarin Coumadin

Administration Subcutaneous or IV Oral

Action Antithrombin effect Vitamin K antagonist

Effect Immediate Slow-acting

Duration Short Long

Test for monitoring APTT PT

Reversed by Protamine sulfate Vitamin K

Other Requires AT-I11 to be effective Decreases production of 11, VII, IX, X

HEMATOLOGY REVIEW 327 I

- - -.

Thrombolytic Therapy

Purpose To activate fibrinolytic system. Used to treat acute myocardial infarction, deep vein thrombosis, pulmonary emboli.

Drugs used Streptokinase, urokinase, tissue plasminogen activator (TPA), pro-urokinase (Pro-UK), acylated plasminogen streptokinase activated complex (APSAC)

Baseline studies PT, APTT, CBC, fibrinogen

Changes induced by therapy in fibrinogen, plasminogen, a-2-antiplasmin, factors V, VIII, IX, XI, XII. I' in plasmin, fibrin(ogen) degradation products, D-dimer, APTT, PT, thrombin time.

Precautions Limit venipunctures. Apply pressure following venipuncture to stop bleeding.


- -

-

Sources of Error in Coagulation Testing

Error Comment

Incorrect anticoagulant 3.2 % or 3.8 % sodium citrate should be used. Labile factors are preserved better. 3.2% is preferred. Less subject to effects of polycythemia.

Drawing coagulation tube Contamination with other anticoagulants can interfere. before other anticoagulant tubes

Probing to find vein Tissue thromboplastin activates coagulation and shortens times.

Incorrect ratio of blood Need nine parts of blood to one part of anticoagulant. Tubes less than 90% full to anticoagulant will have longer times.

Failure to mix anticoagulant Blood will clot. with blood

Polycythemia Hematocrits above 55% lead to longer times. Anticoagulant must be reduced.

Heparin contamination from Will prolong times. Lines must be flushed with saline and the first 5 mL of blood catheter or heparin lock drawn discarded.

Hemolysis Hemolyzed RBCs may activate clotting factors. Hemolysis may interfere with pho- toinetric reading.

Lipemia May interfere with photo-optical methods. May be tested with electromechanical instrument.

Continued


Sources of Error in Coagulation Testing Continued

Error Comment

Improper storage of specimen Should remain capped to prevent change in pH. Test within 2 hours if stored at room temperature or 4 hours if stored at 4°C. Loss of labile factors will prolong times.

Improper storage or Run controls once each shift to verify system performance. reconstitution of reagents

Equipment malfunction, Run controls once each shift to verify system performance. e.g., temperature, timer, detector, volumes dispensed


- - -A-

Causes for Rejection of Specimens for Coagulation Testing

More than 2 hours at room temperature

Exposure to extremes of temperature

Tube less than 90% full

Specimen clotted

Specimen hemolyzed


- - - --- - -- -

- - -

Immunology Terms

Acute phase reactant Protein that increases due to infection, injury, or trauma (e.g., C-reactive protein, alpha-1 antitrypsin, haptoglobin, fibrinogen, ceruloplasmin, alpha-1 acid glycoprotein, complement)

Alloantibody Antibody formed in response to antigens from individuals of the same species

Antigen A foreign substance that stimulates antibody production. Large, complex molecules (MW >10,000), usually protein or polysaccharide.

Antibody Immunoglobulin produced by plasma cells in response to an antigen

Autoantibody Antibody against self

Avidity Strength of bond between antigen and antibody

Chemotaxin Chemical messenger that causes migration of cells in a particular direction

Clusters of Antigenic features of leukocytes differentiation (CD)

- - - - -

Cytokine Chemical messenger produced by stimulated cells that affect the function of other cells

Epitope Determinant site on an antigen

Hapten A low molecular weight substance that can bind to an antibody once it is formed, but that is incapable of stimulating antibody production unless it is bound to a larger carrier molecule

Continued

334 IMMUNOLOGY REVIEW

- -- -- -- A

-

Immunology Terms Continued

Histamine A vasoactive arnine that is released from mast cells and basophils during an allergic reaction

Hypersensitivity A heightened state of immune responsiveness that causes tissue damage in the host

Immunity Resistance to infection

Immunogen Any substance that is capable of inducing an immune response

Immunoglobulin Antibody

Immunology The study of the reactions of a host when exposed to foreign substances

Inflammation Cellular and humoral mechanisms involved in the reaction of the body to injury or infection

Interferons Cytokines produced by T cells and other cells that inhibit viral synthesis or act as immune regulators

Interleukins Cytokines produced by T cells or macrophages that stimulate a number of cell types

Ligand A molecule that binds to another molecule of a complementary configuration. The substance being measured in an immunoassay.

Lymphokines Polypeptides given off by antigen-stimulated T cells, which regulate the function of other cells

Lysozyme Enzyme found in tears and saliva that attacks cell walls of microorganisms

Continued

IMMUNOLOGY REVIEW 335

- -

- --. -

Immunology Terms Continued

- - p p p p - -

MHC Major histocompatibility complex. Genes that control the expression of proteins found on all nucleated cells.

Monoclonal antibody An antibody derived from a single B-cell clone

Opsonin - - -

Serum proteins that attach to a foreign substance and enhance phagocytosis

Phagocytosis The engulfment of cells or particulate matter by neutrophils and macrophages

Plasma cells Transformed B cell that secretes antibody

Polyclonal antibody Antibody produced by many B-cell clones

Postzone False-negative reactions in a serological test due to antigen excess

Prozone False-negative reactions in a serological test due to antibody excess

Seroconversion The change of a serological test from negative to positive due to development of detectable antibody

Serum sickness A type 111 hypersensitivity reaction that results from buildup of antibodies to animal serum used in some passive immunizations

Thymus A small, flat bilobed organ found in the thorax. Site of T-lymphocyte development.

Titer A means of expressing the concentration of an antibody. The reciprocal of the highest dilution in which a positive reaction occurs.

Vaccination Injection of immunogenic material in order to induce immunity


Branches of the Immune System

Branch Definition Defense Against Cells Involved Examples

Cellular Cell mediated Viruses, fungi, myco- T lymphocytes bacteria, other intracellular pathogens, tumor cells

Graft rejection, hypersensitivity reaction, elimination of tumor cells

- - - - - --

Humoral Antibody mediated Bacteria (extracellular) B lymphocytes, plasma cells Antibody production


'Ifrpes of Immunity

n'pe Explanation Components Memory?

Natural or innate Nonspecific defenses with Intact skin, mucous membranes, stomach No which one is born acidity, cilia, mucus, skin secretions,

lysozyme, IgA, phagocytes, complement, interferon, inflammation

Acquired or adaptive Specific responses to T cells, B cells, plasma cells, antibodies, Yes eliminate a microorganism cytokines


--- - -

Adaptive Immunity

VPe Explanation Example Specific? Immediate? Long-Term?

Naturally acquired An individual infected Clinical or subclinical Yes No Yes active immunity with a microorganism infection

produces an antibody.

Artificially acquired An individual exposed DPT, MMR, polio, Yes No Yes active immunity to an antigen through a tetanus, Haemophilus

vaccine develops influenzae type b immunity without having vaccine the infection.

Naturally acquired An individual is pro- Babies are protected by Yes Yes No passive immunity tected by antibodies pro- maternal antibodies that

duced in another cross the placenta and individual. are present in breast

milk.

Artificially acquired An individual receives Rh immune globulin, Yes Yes No passive immunity an immune globulin HBIG, anti-toxins

containing antibodies produced in another individual.


- -- -- - -.-

--- -- --

Cells of the Immune System

Cell Function Comments ---- ~~ ~ ~ - - - - - - - - - -

T lymphocytes Cell-mediated immunity - - - - - - --

Derived from cells in bone marrow. Develop T-cell specific surface antigens in thymus (e.g., CD2, CD3, CD4, CD8). 60-80% of peripheral lymphocytes.

Helper/ Orchestrates cell-mediated immunity. CD4+ (T4). 55-70% of peripheral T cells. Normal inducer T cells Activates B cells, cytotoxic cells, and CD4 = 1,00O/pL. 111 AIDS, less than 200/bL.

natural killer cells

Suppressor/ Suppressor cells inhibit helper T cells. CD8+ (T8). 2 5 4 0 % of peripheral T cells. cytotoxic T cells Cytotoxic cells kill other cells. (Normal CD4:CD8 ratio is 2:l. In AIDS, less than

0.5:l).

B lymphocytes Synthesize and secrete immunoglobulins Develop in bone marrow. Surface immunoglobu- after antigenic challenge lins and CD19, CD20, CD21. Less than 15% of

lymphs.

Natural killer Recognize and destroy virally infected cells Non-T, non-B. Lack CD3, CD4, and CD8. (NK) cells and tumor cells (null cells)

Lymphokine- Use IL-2 to help lyse tumor cells Non-T, non-B activated killer cells (LAK)

- ~ ~ ~ ~ - - - - - -

Continued


Cells of the Immune System Continued

Cell Function Comments

Eosinophils Suppress inflammatory reaction. Kill some Some phagocytic ability parasites.

Basophils Granules contain histamine and heparin. Some phagocytic ability. Precursor of mast cell. Role in hypersensitivity reactions.

Neutrophils Principal leukocyte associated with phagocytosis and localized inflammatory response. Granules contain bactericidal enzymes. Respond to chemotaxins.

Monocytes Phagocytes. Respond to chemotaxins. Pro- Precursor of macrophage cess antigens and present to lymphocytes. Produce interleukin-1 and other cytokines.

1MMUNOLOGY REVIEW 341

- - -

-- - -

Laboratory Identification of Lymphocytes , To Obtain Lymphocytes

Density gradient centrifugation with Layers from top to bottom: plasma, mononuclear cells, Ficoll-Hypaque, Ficoll-Hypaque RBCs and granulocytes

To Identify Lymphocytes

Direct or indirect immunofluorescence Use of labeled monoclonal antibodies against specific surface antigens. Slides are read with a fluorescent microscope.

Flow cytometry (FACS: fluorescence- Use of labeled monoclonal antibodies against specific surface antigens. activated cell sorter) Light scattering is measured as cells flow through a laser beam.

Surface immunoglobulins Immunoenzymatic method to demonstrate immunoglobulins on B cells

Rosette technique T lymphocytes form rosettes with sheep RBCs. Old method.


-- - -

Common Lymphocyte Markers

T Cells B Cells

- - - -

Surface immunoglobulins


Immunoglobulin Structure

Basic structure A four-chain polypeptide unit that consists of two heavy chains and two light chains held together by disulfide bonds

Heavy chains y, a, p, 6, and E. The two heavy chains in the immunoglobulin molecule are always of the same type. They determine the immunoglobulin class (IgG, IgA, IgM, IgD, and IgE).

Light chains K or A. Both K and X light chains are found in all classes of immunoglobulins, but only one type is present in a given molecule.

Fab fragment Fragment antigen-binding. Consists of one light chain and one-half of a heavy chain. The intact immunoglobulin has two Fab fragments, each representing one antigen-binding site.

Fc fragment Fragment crystalline. The carboxy-terminal halves of the two heavy chains. This portion of the molecule has no antigen binding ability.

Constant region The carboxy-terminal end of the immunoglobulin molecule, where the amino acid sequence is the same for all chains of that type.

Variable region The amino-terminal end of the immunoglobulin molecule, where the amino acid sequence varies. This part of the molecule is responsible for the specificity of a particular immunoglobulin. Also known as the antigen-recognition unit.

Hypervariable Regions within the variable region that actually form the antigen-binding site. Through changes region in the hypervariable region, an immense diversity of antigen-binding sites can be created.

Hinge region The flexible portion of the heavy chain, located between the first and second constant regions. This allows the molecule to bend to let the two antigen-binding sites operate independently.

~ - - - - - - - - - ~~~~ ~-~ - - ~ - - - - ~

Joining chain A glycoprotein that serves to link immunoglobulin monomers together. Only found in IgM and se- cretory IgA molecules.


- - - - - - - - - -- --- - -- - --

Immunoglobulins

Form Monomer Pentamer Monomer and dirner Monomer Monomer

Molecular weight 150,000 900,000 160,000 or 400,000 180,000 190,000 (daltons)

Heavy chain Gamma (y) Mu (14 Alpha (4 Delta (6) Epsilon ( E )

Light chain Kappa or K O ~ X K or h K or h K or A lambda (K or X)

-

% of total immuno- 75-80% globulin

Serum concentration 800-1600 120-1 50 70-350 mg/dL 1-3 mg/dL 0.005 mg/dL mg/dL mg/dL

Antigen-binding sites 2 10 2 or 4 2 2

Complement fixation Yes Yes No No No - ~ p p

Crosses placenta? Yes


Continued

- - - --

Immunoglobulins Continued

IgG IgM I@' I sE

Other Defense against bacteria First Ig produced In tears, sweat, Function Role in allergic reactions. and viruses. Neutralizes in an immune saliva, respiratory unknown Binds to mast cells and toxins. Opsonin. More response. Only and GI mucosa. triggers degranulation efficient at precipitation Ig produced by First line of and release of histamine than agglutination. newborn. Neu- defense. and heparin.

tralizes toxins. Opsonin. Most efficient Ig at initiating coln- plement cascade. More efficient at agglutination than IgG. Destroyed by sulfhydryl compounds.


- - -

Complement

Definition A series of more than 25 serum proteins that interact to enhance host defense reactions. Most are inactive enzyme precursors that are converted to active enzymes in a precise order (cascade).

Functions Opsonization, chemotaxis, cell lysis

Classical pathway Triggered by antigen-antibody reactions. IgM is most efficient activator. A single molecule attached to two adjacent antigenic determinants can initiate the cascade. IgG1, 2, and 3 can activate complement but a t least two molecules are required. Recognition unit: C1 (first to bind). Activation unit: C4, C2, C3. Membrane attack complex: C5, C6, C7, C8, C9 (drills holes in cell membrane).

Alternative pathway Activated by bacteria, fungi, viruses, tumor cells, some parasites

Present in highest C3 concentration in plasma

Key component of both C3 pathways

Ions required Calcium, magnesium

Inactivation 5G°C for 30 minutes

Clinical manifestations Increased susceptibility to infection or accumulation of immune complexes with of deficiency autoimmune manifestations

Significance of decreased levels Immune complex disease


Immune Phenomena - - - -

Explanation Application

Primary phenomena Combination of antibody with antigen Not easily detectable. Can be measured indi- rectly by radioimmunoassay (RIA), enzyme immunoassay (EIA) , immunofluorescence.

Secondary phenomena Precipitation, agglutination, Measured more readily. Basis for many complement fixation serological tests.

Tertiary phenomena In viva reactions; e.g., inflammation, Some are useful diagnostically, e.g., skin phagocytosis, deposition of immune testing. complexes, immune adherence, chemotaxis


-

Hypersensitivity Reactions

m e 111: I)rpe IV: Q p e I: Anaphylactic I)rpe 11: Cytotoxic Immune Complex Cell Mediated

Key reactant(s) IgE IgC, JgM, complement, IgG, IgM, complement T cells and antigens found on macrophages cell surfaces

Result Release of mediators Cytolysis due to anti- Deposits of antigen- Release of from mast cells and body and complement antibody complexes lymphokines from basophils antigen-stimulated

T cells

Symptoms Immediate Immediate Immediate Delayed

Example Anaphylaxis, hay fever, Bansfusion reactions, Serum sickness, Contact dermatitis, asthma, food allergies hemolytic disease of systemic lupus tuberculin test

the newborn, auto- erythematosus (SLE), immune hemolytic rheumatoid anemia, idiopathic arthritis (RA) thrombocytopenic purpura (ITP), Good- pasture's syndrome

350 lMMUNOLOGY REVIEW

-- --- - -

Agglutination Methods

Method Principle Examples

Direct agglutination The process by which particulate antigens, such as Febrile agglutinins, Salmonella cells, aggregate to form large complexes when and Shigella serotyping specific antibody is present

Hemagglutination An antigen-antibody reaction that results in the ABO slide typing clumping of RBCs

Passive agglutination A reaction in which soluble antigens are bound to Rheumatoid factor latex beads, bentonite, or charcoal. The particles are agglutinated by the corresponding antibody.

- - -

Passive hemagglutination A reaction in which soluble antigens are adsorbed Cold agglutinins onto RBCs. The RBCs are agglutinated by the corresponding antibody.

Reverse passive A reaction in which carrier particles coated with Rapid tests for identification of agglutination antibody clump together due to combination with bacteria

antigen - Agglutination inhibition An agglutination reaction based on competition Slide tests for human chorionic

between particulate antigen (reagent) and soluble gonadotrophin (HCG) antigen (specimen) for limited sites on a reagent antibody. Lack of agglutination is a positive result.

Continued


-- - -

-- -

Agglutination Methods Continued

Method Principle Examples

Hemagglutination A test for detecting antibodies to certain viruses that Rubella antibody inhibition agglutinate RBCs. In the presence of antibody, the

virus is neutralized and hemagglutination does not occur.

Coagglutination An agglutination reaction in which bacteria are used as Rapid tests for identification the carrier for the antibody. (Protein A on the surface of of bacteria S. aureus adsorbs the Fc portion of the antibody molecule.)

Rheumatoid factor can cause false-positive reactions in agglutination tests because it reacts with any 1gG. Heterophile antibodies can cause false-positive reactions in hemagglutination tests.


- - - --

Precipitation Methods - -

Method --

Principle Application

Precipitation Soluble antigen combines with soluble antibody to pro- See following examples. duce visible insoluble complexes. Precipitation methods are less sensitive than agglutination methods because more antibody is needed to produce a visible reaction.

Flocculation Soluble antigens react with specific antibody to form a VDRL, rapid plasma reagin (RPR) precipitate of fine particles.

Ouchterlony Antigens and antibodies diffuse out from wells cut in gel Fungal antigens, extractable technique and form precipitin lines where they meet. Double nuclear antigens

immunodiffusion.

Counter immuno- Antigens and antibodies are placed in wells that are Bacterial antigens electrophoresis directly opposite one another in a gel. An electrophoretic (CIE) charge is applied to drive the reactants toward each other.

A precipitin band forms where they meet.

Immunoelectro- Proteins are separated by electrophoresis, then subjected Identification of immunoglobu- phoresis (IEP) to double diffusion with reagent antibodies placed in a lins in monoclonal

trough cut in the agar. The shape, intensity, and location gammopathies of the precipitin arcs that develop are compared with those of a normal control.

Continued


- -- - - - - pp - . - -

-

Precipitation Methods Continued

Method Principle Application

Immunofixation Proteins are separated by electrophoresis. A cellulose Identification of immunoglobu- electrophoresis (IFE) acetate strip impregnated with antiserum is placed on the lins in monoclonal

separated proteins. The antiserum diffuses into the gel gammopathies and antigen-antibody complexes precipitate.

-

Radial immuno- Antigen is measured based on the diameter of a precipitin Quantitation of immunoglobulins diffusion (RID) ring that forms when it diffuses out of a well in a gel- and complement

containing antibody. - - - - - - - - - - - - - -

Rocket electro- An electrical charge is applied to an RID assay. The height Immunoglobulins, complement phoresis of the rocket-shaped precipitin band obtained is propor-

tional to the concentration of the antigen.

Nephelometry Light scattering by immune complexes is measured. Immunoglobulins, complement, C-reactive protein


- -- - - --

Other Serological Methods

Method Pn'ncivle Comments Examvle

Complement fixation Patient serum is incubated Patient serum must he inacti- Viral, fungal, rickettsia] with antigen and complement. vated. Best for demonstration antibodies If the corresponding antihody of IgM antibodies. More sensi- is present in the serum, it tive than agglutination and pre- forms a complex with the anti- cipitation, but less sensitive gen and complement. When than labeled immunoassays. sensitized RBCs are added, Not widely used. there is no free complement to lyse them. No hemolysis is a positive reaction.

Direct fluorescent The specimen is placed on a Detects antigens. Fluorescent Bacterial antigens antibody glass slide and overlaid with compounds absorb energy from

fluorescein-labeled antibody. light source and convert it into If the corresponding antigen light of a longer wavelength is present, the labeled- (lower energy) than that antibody binds and fluores- absorbed. Fluorescent labels: cence will be seen with a fluorescein isothiocyanate or fluorescent microscope. rhodamine B isothiocyanate.

Continued


Other Serological Methods Continued

Method Principle Comments ---

Example

Indirect fluorescent antibody

Reagent antigen on a glass "Sandwich technique." Detects Fluorescent antinuclear slide is overlaid with patient antibodies in serum. antibody (FANA), serum. If the corresponding fluorescent trepone~nal antibody is present in the antibody (FTA) serum, it attaches to the antigen. When fluorescein- labeled antihuman globulin is added, it attaches to the antibody. Fluorescence will be seen with a fluorescent microscope.


Labeled Immunoassay Terminology

Ligand The substa~lce being measured in an imlnunoassay

Isotopic Ail immunoassay that uses a radioisotope as the label - - - - - - - -

Nonisotopic An imniunoassay that uses something other than a radioisotope as the label; e.g., enzyme, fluo- rochrorne, chemiluminescent molecule

-

Competitive An irnniu~loassay in which the patient ligand and the labeled reagent ligand compete for a lirn- ited number o f binding sites on a reagent antibody

-

Noncompetitive An irnmunoassay in which the reaction does not involve competition for binding sites. Noncom- petitive assays Jre more sensitive than coiiipetitive assays.

-

Heterogenous A11 ~~nn i i~noassay in which a separation step is requlred to remove free reactant from bound reactant. Heterogeneous assays are inore sensitive than liomogeneous assays.

Ho~nogeneous An i~nmunoassay in which a separation step is not required. Hon~ogenous assays are easier to automate.


Comparison of Radioimmunoassay and Enzyme Immunoassay

--

RIA EIA

Label Radioisotope, usually 1251 Enzyme

Measurement Counts per minute in sci~ltillation counter Color develop~nent after addition of substrate

Advdiitages Sensitivity Specificity

Sensitivity Specificity Long sheli-life of reagents No radiation hazard No special disposal requirements No need for scin~illation counter

Disadvailtages Short shelf life of leagents Radiation hazard Regulations governing disposal of radioisotopes


Principles of Labeled Immunoassays

Method V p e of Assay Principle

RIA Competitive Radiolabeled ligand and unlabeled ligand in the specimen compete for Isotopic binding sites on reagent antibody. The more unlabeled ligand in the Heteroge~irous specimen, the less labeled ligand binds. Free labeled ligand is separated

from bound labeled ligand. Solid-phase attachment is the most frequent separation method. For example, antibody is adsorbed to the inner wall of the assay tube and separation is achieved by aspiration or decanta- tion. The amount of labeled ligand bound is determined by the counts per minute (CPM) 011 a scintillation counter. CPM are inversely proportional to the concentration of the ligand in the specimen.

! Imniu~loradiometric Noncompetitive The specimen is incubated with a labeled antibody. Solid-phase ligand assay (IRMA) Isotopic is added to remove unbound labeled antibody. After centl-ilugation, the

Heterogeneous radioactivity in the supernatant is determined. CPM are directly propor- 1 tional to the concentration of ligand in the specimen. I

Enzyme-linked im~nunosorbent assay (ELISA)

Competitive Enzyme-labeled ligand and unlabeled patient ligand compete for hind- Nonisotopic ing sites on antibody niolecules attached to a solid phase. Free labeled Heterogeneous ligand is removed by washing and a substrate is added. The enzyme ac-

tivity is inversely proportional to the concentration of the ligand in the specimen.

Irldirect or non- Nonco~npefitive Antibody in the specimen attaches to a solid-phase antigen. After incrl 1 competitive ELlSA Nonisotopic batiori and washing to remove u ~ ~ h o u n d antibody, an enzyme-labeled I

Heterogeneous antiglobulin is added. This second antihody reacts with the Fc portion I of the patient antibody bound to the solid phase. Following another I

I Continued I

360 IMMUNOLOGY REVIEW I

Principles of Labeled Immunoassays Contirlued

Method Q p e of Assay Principle

Substrate-labeled Col~lpetitive Unlabeled ligand in the specimen ' ~ n d fluorogenic Iigand compete for fluorescent Nonisotopic sites on reagent antibody. Free Idbeled ligand produces tluorescence. immunoassay Homogeneous Bound labeled ligand does not produce fluorescence. Fluorescence is (SLFLA) directly proportional to the concentration of the ligand in the specimen.

This is an automated neth hod.

Fluorescence Competitive Unlabeled hgand in the specimen and fluorogenic ligand compete for polarization Nonisotopic sites on reagent antibody. Free labeled ligd~id rotates rapidly and emits imrnunoassay (FPIA) Homogeneous little polarized tluorescence. Bound labeled ligand rotates more slowly

and emits more polarized fluorescence. The higher the concentration of bound labeled ligand, the Illore poldrized fluorescence. The amount of polarized fluorescence is iilversely proportional to the concentration of the ligand in the specimen. This is an automated method.


Nontreponemal Tests for Syphilis

VDRL RPR

Method Flocculation Flocculation

Detects Reagin Reagin

Reagent (s) Cardiolipin Cardiolipin with charcoal added

Positive reaction Microscopic clumps Macroscopic agglutination

Specimen(s) Inactivated serurn, CSF Serum (inactivation not required), plasma

Reactivity during May be negative in primary stage. Titers Sarne as VDRL disease usualIy peak during secondary or early

late stages. Titers decline in late stage, even when untreated. More rapid decline with treatment. Becomes nonreactive following successful treatment.

False-positives 10-30% may be biologic take positives; Sarne as VDRL e.g., infectious niononucleosis (IM), infectious hepatitis, malaria, leprosy. lupus erythematosus, rheumatoid arthritis, advanced age, pregnancy. Positive in other treponemal infections such as yaws and pinta.

Other Replaced by RPR for serum. Still performed Good tor screening and treatment monitoring 1 on CSF.

364 IMMUNOLOGY REVIEW I

Treponemal Tests for Syphilis

FTA-ABS Microhemagglutination Assay

Metllod lndirect fluorescent antibody Hemagglutination

Detects Ailtibody to T pallidurn Antibody to T pallidrlrr~

Reagent (s) Sorbent (nonpathogenic treponemes-Reiter Sheep RBCs coated with antigens from strain), slides with Nichols strain of T pulli- Nichols strain of T pallidiirn, sorbent dnm, fluorescein-labeled antihu~nan globulin

Positive reaction Fluorescence Rough or jagged pattern of cells in microtiter wells

Specimen(s) Serum, CSF Serum

Reactivity during Usually positive before reagin tests. Some Not as sensitive in primary syphilis as disease false-negatives in primary syphilis. Usually FTA. Sensitivity close to 100% in sec-

positive for life. ondary syphilis. Usually positive in late stages.

False-positives Fewer than nontreponemal tests. Reactivity is Fewer than nontreponemai tests. seen with other trepone~nal diseases, notably yaws and pinta.

Other Sorbent rernoves nonspec~fic antibodies. Sorbent reliloves nonspecific antibodies. Used Used to confirm reactive nontreponemal test. to confirm reactive nontreponemal test. Not Not good for treatment monitoring. good for treatment monitoring.

IMMUNOLOGY REVIEW 365 ~

Tests for the Diagnosis of Infectious Mononucleosis (IM)

Antibodies Interpretation I I

I Heterophile antibodies* I I

1M antiboclies: Agglutinate sheep, beef, ox, and horse RRCs. Adsorbed by beef RBCs but not by guinea pig kidney cells (GPK).

Serum sickness antibodies: Agglutinate sheep, beef, and horse RBCs.

Adsorbed by beef RBCs and guinea pig kidney cells. Forssman antibodies: Agglutinate sheep and horse

RBCs. Adsorbed by guinea pig kidney cells but not by beef RBCs.

Agglutinalion of sheep or horse cells following adsorption with GPK but riot with beef RBCs = IM.

No agglutination ot sheep or horse cells following adsorption with GPK or beef KRCs = serum sickness.

Agglutination of sheep or horse cells following adsorption with beef RBCs but riot with GPK = Forssman.

-

Specific viral antibodies

IgM-VCA (viral capsid antigen): Peaks 3-4 weeks after IgM anti-VCA, anti-EA, or IgG anti-VCA without anti- infection. Undetectahlr after 12 weeks. EBNA = recent or current infection.

IgG-VCA: Appears in late acute phase and persists Anti-EBNA or IgG anti-VCA without IgM anti-VCA = past for life. infection.

Anti-EA (early antigen): Appears early and persists for years.

Anti-EBNA (EB nuclear antigen): Appears 2-3 months after infection and persists indefinitely.

*No~ispecit'ic antibodies that cross-react with antigcns other than those originally responsible for (heir prutluction


Weil-Felix Reaction !

Weil-Felix Renction I

Disease Organism TPansmission OX-2 OX-19 OX-K I

Rocky Mountain Rickettsia rirkettsii Dog or wood tick + ++++ 0 spotted fever*

Epidemic typhus R. prouluzekii Body or head louse + ++l+ 0

Eilde~nic typhus R. typhi Rat flea + t + + t 0

Scrub typhus R. tsutsugarr~ushi Mite 0 0 ++++ Q fever CoxieLLo burnetii Inhalation of barnyard dust or 0 0 0

ingestion of infected meat or milk

*Most conilliolr rickettsia1 disease in U.S.

IMMUNOLOGY REVIEW 367 1

Antinuclear Antibodies I

Indirect Fluo- I

rescent Antibody Antibody Against Specificity Sensitivity (IFA) Pattern Contrnents

I I

Anti-ds-DNA Double- Specific for SLE Found in only 50- Peripheral or Considered 1 stranded DNA 80% of patients ho~nngeneous diagnostic for

with SLE SLE, especially when C3 is low.

Anti-Sm Extractable nu- Specific for SLE. Onlv found in 30% Speckled clear antigen Not found in of patients with associated with other disorders. SLE RNA

Allti-DNP Deoxyribonu- Present iu SLE Present in 70-90% Peripheral or LE factor i cleoprotein and drug-induced of patients with homogeneous 1

SLE SLE

Anti-SS-A/Ro Extractable Present in SLE, Present in 25-40% Speckled Most often in pa- l~uclear antigen. scleroderrna and of S1.E patients tients with cutan- RNA protein Sjogren's eous manifesta- conjugate. syndrome tions (photosensi- 1

tivity dermatitis) I !

Anti-SS-R/La Ribonucleo- Present in SLE, Present in 10-15 0io Speckled protein scleroderma and of SLE patients I

I Sjogren's syndrome I

I Continued ~


Hepatitis Tests

Test Indication

Hepatitis A

Anti-HAV (total) Past infection and i~lilrlunity

Anti-HAV (1gM) Acute hepatitis A

Hepatitis B

I-IBsAg Acute or chronic infection HRV (carrier), infectivity

HBeAg Acute or chronic HBV (carrier), high degree of infect~vity

Anti-IIBc (total) Current or previous infectiun or a carrier

Anti-HBc (IgM) Recent acute infection

Anti-HBe Disease resolution and reduced infectivity

Anti-HBs Recovery and imlllunity

Hepatitis C

Anti-HCV Acute, chronic, or previous hepatitis C infection

Hepatitis D

Anti-HD Past or cur ren~ hepatitis D infection

Hepatitis E

None


HIV Testing

Test Detects ripe of Test Principle Corrtments

ELISA

-

Slide agglutination tests

Antibody to f1IV Screening

- --

Alltibody to HIV Screcni~ig

Viral ailtigel1 IS coated oil s solid support. Patient serum added After il~cubation and washing, enzyme-labeled c ~ n t i l ~ u ~ n a n globulin (AHG) is added followed by substrate.

f IlV antigen is adsorbed onto carrier particles. The particles agglutinate in the presence of antibody.

Most widely used screening procedure Pos~tiveb must be confimled. Anti-HIV-1 and anti-HIV-2 (or comb~nat io~l test) required on blood donors.

--

Used when instrurrlentnrion is not avallahle.

Western blot Antibcrdy to HIV Confirrrlatory A lysate of HIV dntigen is Most cornnio~~ly used sepdrated into conlponents by confirnmatory test. I I IV inkc- electrophoresis and transferred tion is indicated by reactivity to nitrocrllulose paper. The in twu oi the tol lowi~~g bands: resulting blot is cut into stl-ips p24, gp41, or gpI2U/lhO. and reacted with serum. Ldbefed an t ih i~n~dn globulin is ddded.


Other Serological Tests

Test Common Method Reagents interpretation Comments

Rheumatoid Agglutinatioil IgG attached to Positive in 70-80% Rheumatoid factor is an autoantibody arthritis (RA) latex or RBCs of patients with RA (usually IgM) against IgG. Not spe-

cific for RA. Also found in SLE, scleroderma, Sjogren's syndrome, B-cell lymphoproliferative disorders.

Cold Hemagglutination Patient or Positive in primary PAP is caused by Mycoplusrna prreu- agglutinins Group 0 RBCs atypical pneumonia moniur. Illcreased titer of Anti-I. Test

(PAP) is incubated in refrigerator. Specime~r must be kept warm prior to testing to prevent fdlse-negatives or decreased riters. l-'ositives must be confirmed by reversal of agglutination at 37°C.

Anti-strep- Neutralization Streptolysiii 0 , Titer is highest Streptolysin 0 lyses RBCs. Anti-streptolysin group O KBCs dilution showing tolysin 0 in patient's s e n ~ n ~ neutral- 0 (ASOJ no hernolysis. High izes streptolysin 0 reagent so RBCs

titers with strep are not lysed. Streptolysin 0 is oxy- infecrions, rheu- gen Idbile. RBC control (RRCs + matic fever, glo- buffer) should show no hemolysis. ~rierulonephritis. Streptolysin control (streptolysi~~ 0 +

buffer + RBCs) should show hrmoly- sis. Reported in Todd units (reciprocal of highest dilution showing no hemolysis). Norrnal is less than 166.

Continued

374 TMMUNOLOGY REVIEW

SECTION 6

Immunohematology Review

Primary vs. Secondary Response

Primary Secondary (Anamnestic)

Stirnulus First exposure to antigen Subsequent exposure to antigen

Lag Phase Days to months Hours

Type of antibody IgM at first. May switch to IgG after 2-3 weeks IgG (isotype switching).

Titer Rises slowly. Peaks then declines. Rises faster and higher. Stays elevated longer.

380 IMMUNOHEMATOLOGY REVIEW

IgG vs. IgM

Structure Monomer Pcntarner

Number of antigen-binding sites 2 10

'&pe of antibody Itnn~une Naturally occurring

Opt i~n~un temperature of reactivity 37°C 25°C or lower

Reacts in saline No Yes --

Reacts best by indirect antiglobulin test (IAT) Yes No

Complement fixation Moderare Strong

Causes tr~nsfusuon reactions Ties hot usually, except ABO

-- - -

Crosses placenta Yes No

Causes helnolytic disease of the ~iewborn Yes No

Destroyed by sl~lfhyclryl compounds (dithiothrcitol [DTT], No Yes 2-n~ercaptoethanol [2-ME])

-

IAllMUNOI-IGMATOLOGY REVIEW 381

Signs of Antigen-Antibody Reactions in Blood Bank Testing

Explanation Union of antibody with antigen activates Antibodies attach to antigens on adjacent complement which destrvys RBC membrane. RBCs, forming bridges between cells. Requires CaL+.

!

Immunoglobulins One IgM n~ulecule or two IgG molecules can IgM is more effective in inducing agglutina- !

activdte complement. (Kequires two Fc portions tion because of its size and number of anti- of immunvglnhulin in close proximity on the gen-binding sites. I RBC surface.)

Grading PH = partial hemolvsis; some RBCs remain mf = rnixed field (some agglutinated RBCs I

H = complete hemolysis, no RBCs remain in sea of free RRCs) !

w i = tiny agglutinates, turbid background I 1 + = small agglutinates, turbid hackground 2 + = niediurn-sized agglutinates, clear

background 3+ = several large agglutinates 4+ = orie solid agglutinate

Comments False-negative reaction if overlooked. Most common reaction seen.

Factors that Affect Agglutination

Factor Conlrrlents

Sensitization Stage Attachment of antibody to antigen

Temperature Cli~iically significant antibodies react best at 37°C.

PH Most antibodies react at plI 5.5-8.5

Ionic strength Reducing the ionic strength of the medium facilitates interaction of antibody with antigen (e.g., low ionic strength solutiori [LISS]).

A1ltigen:antibody ratio Too r~luch antibody can cause prozone (ialse-negative). Optirnul~l serum to cell ratio is 8O:l. Usually two drops of serum to one drop of 2-5% RRCs. Follow the nianufacturer's directions.

Incubation t ~ m e Depends on nledium. Usually 10-30 minutes. Follow the ma~~ufacturer's directions.

Agglutil~ation Stage Formation of antigen-antibody bridges between RBCs

Type oi antibody molecule 1gM is larger and can span the distance betweell RBCs more easily

Density of antigens and Affects ease of attach~nent of antibodies location or] RBC surface

Zeta potential The difierence in charge between the riegatively charged RBC surface and the cloud of positive ions that surround the RBCs. Reducing the zeta potential allows the RBCs to move closer together (e.g., enzyme treatment of test cells).

IMMUNOHEMATOLOGY REI'IEW 383

Antigen-Antibody Enhancement

Albumin 22% or 30% bovine serum alhui~iin. Reduces net negative charge of RBCs, allowilig thern to come closer toget her.

Low ionic Lowers ionic strength of st~spending medium, allowing antigens and antibodies to move closer strength together more rapidly. Reduces the incubation time in the indirect antiglobulin test to 10 solution (LISS) rninutes.

Polythylenegiycol Increases antibody uptake. Used for detection and identification oi weak IgG antibodies. (PEG)

Enzymes Exarnples include ficin. bromelin, papain. Reduce R R C surface charge by cleaving sialic acid molecules. M, N, S, Fyd, and Fyh antigens are destroyed.


Antihuman Globulin Serum

Q P ~ Detects Comments

Polyspecific (broad spectrum) IgG and C3d

Monospecific

Anti-IgG

Used for direct antiglobulin test (DAT) and, in some labs, for routine compatibility tests and antibody detection. If positive, monospecific sera may be used for follow-up testing. Disadvantage: reactions due to complenlent binding by clinically insignificant cold antibodies.

Can be used for routine compatibility tests and antibody identification. Detects clinically significant antibodies.

Anti-C3d and antiLC3b C3d and C3b* Helpful in investigation of immune hemolytic anemia

'Complement proteins.

IMMUNOHEMATOLOGY REVIEW 385

Antiglobulin Testing

Direct (DAT) Indirect (lAT]

Detects In-viva sensitization of RBCs by In vitro sensitization of RBCs by IgG antibody IgG antibody

Specimer~ EDTA (ethvlenediarninetetra-acetic acid) Serum, plasma, RBCs red cells preterred

Incubation None required Serum or plasma with reagent RRCs or KBCs with reagent antiserum

Application Ilemolytic disease o i the newborn. Antibody screen, crossmatch, RBC phenotyping, transiusion reactions. autoin~mune weak D testing (D2) hemoiytic anemia, dr-11g-induced hemolytic anemia

False-positives Complement binding in vitro i f RBCs are Cells wit11 positive DAT. t<lken from '3 red-top tube and broad- Ovel-centrif~~gation. spectrum antihum,m globulin ( A X ) is ~rsed.

Septicemia. Contarnination of specimen. \Vharton's jelly in cord blood. Overreading. Overcentrifi~gation.

Continu~rf


Frequency of ABO Types

m v e Whites (%I Blacks (%) Hbvanics (%I Asians (%)


ABO System

Group Antigen(s) on RBC Serum

0 Nei ther Anti-A, Anti-B

AB A and B Neither ant i-A n o r anti-B

392 IMMUNOIIEMATOLOGY REVIEW

ABO Discrepancies

Anti-A Anti-B A , cells A, cells B cells 0 cells Auto Possible Cause* Resolution

0 0 0 0 0 0 0 Missing isosgglutinins Repeat reverse grouping in group 0 using four drops of

serum. Iricubate at room temperature for 30 minutes or at 4OC. (Run auto control and screening cells.)

4 + 0 1 + 0 4 + 0 0 A, with anti-A, Type cells with Anti-A, Test serum with additional A, and A2 cells.

4 + 4 + 2 + 2 + 2 + 2+ 2 + Rouleaux Use washed RBCs suspended in saline for forward grouping. Perform saline replacement technique in reverse grouping.

3 + 4 + 1 + 0 0 0 0 A,B with allti-A, Type cells with Anti-A, Test serum with additional A, and Al cells.

"Othei explanations m a y be possible.

Continued


Non-Group Specific Transfusions

Patient v p e

RBC v p e Patient Plasma v p e Patient Can Receive Can Receive

0 0 only 0 , A, B, AB

AB AB, A, B, 0 AB


Using Punnett Square to Predict Rh Qpe

Father's Gerrotype

Mother's Genotype


Frequency of Rh Antigens

A n t i m Whites (%) Blacks (%)

Note: Students do 11ot need to memorize all frequeilcres but should know f~.cquency of D and which antigens are most and least com1n011.


1 Positive Rh Controls I

Possible Cause Resolution

Cells with positive DAT Type with monoclonal anti-D, chemically modified anti-D, or I saline anti-D. !

I Kouleaux or higll-titered cold agglutinin Repeat using washed, saline suspended RBCs. I

1 Bacterial contamination of cells or reagents Repeat with new specimen or reagent.


Rh Typing Sera

High-Protein Chemically Modified Anti-D Monoclonal/ Aati-D Anti-D PolycIonal Blend Saline Anti-D

Description Polyclonal IgG anti-El IgG allti-D converted to Blend of IgM monoclorial IgM ariti-D susprrided in 20-2491 protein direct agglutinin. In 6 % anti-D and IgG polyclonal in saline

protein concentra!ion. anti-D. Low protein content.

-- --

Protein ~ o r ~ - High Low l.onr Low centration - Use Routir~e typing When Rh control is When Rh i:oritrol is When Rh control is

positive with high- positive with high- positive with high- protein reagent protein reagent protrin reagenl

-. - Cor~trol Sarne ingredients a s ABO forward grouping ABO forward grouping 6% bovine albumir~

reagent, except no serves as control for all serves as control for all anti-D. Sho~rld be except type AB. For type except type AB. For type purchased fro111 AB, dn autocol~trol, 6% AD, an auto-control, same manufacturer albumin, or saline may 6% albumin, or saline as Anti-D. be recommended. may be recommended.

Follow the manu- Follow the manufacturer's instructions. facturer's instructions.

-- Used for l'e s Yes Yes No weak L)

testing! -

Cr~ntinued

1MMUNOHEMATOI.OGY REVIEW 405

Selection of Rh Type

Recipient 'Ijpe Rh Q p e Patient Can Receive

Rh positive Rh positive or Rh negative

Weak D Rh positive or Rh negative

R h negative Rh negative or~ly, especially woinen of childbearing age. (If Kh positive must be given in an emergency, Rh immune globulin can be given to prevent immunization.)

IMMUNOHEMAI'O1,OGY REVIEW 407

Z antigen i antigen

Adult cells Much Trace

Cord cells Trace Much

i adult cells Trace Much


Antibody Identification

Reactions Possible Explanation

Same strength and in onc phasr onlv Suggestive of s~ngle antibody

Varying strength Multiple antibodies, antibody exhibiting dosage, antigens of differing strength

In diiferent phases Combination of warm and cold antibodies, antibody with wide thermal range

-.

All cells in AHG, ~utocontrol negative Multiple antil~odies, antibody to high frequency antigen I

All cells in AHG, autocontrol positive Warm autoantibody !

All cells at 37"C, negative in AIIG. autocontrol positive Kouleaux

IbIMUNOHEMATOLOGY REVIEW 413

Cold Antibodies

Anti-A, Only fou~ld in subgroups of A. Agglutinates A, and A,B cells, but not A2 or 0 .

Anti-I Agglutinates all adult cells, except i adult. Does not agglutinate cord cells

Anti-i Agglutinates cord cells more strongly than adult cells.

Anti-H Most comrnon in A, and AIB. Agglutinates O cells most strongly, followed by A, and B; then A, and A,B.

Anti-IH Most conlrllon in A, and AIB. Agglutinates cells that possess both I and H. Agglutirlates adult 0 cells most st~ongly. Wealtcr reactions with A, cells. Does not agglutinate cord cells.


Compatibility Testing Specimen collected within 3 days of transfusion if the patient has been pregnant or transfused during the preceding 3 months.

Confirmation of identifying information on request fnrm and specimen.

Check of blood bank records

Repeat ABO type on donor.

Repeat Rh type on donor if unit 1s labeled R h negative (weak D not required)

ABO type on recipient.

Rh type on recipient (weak D not required).

Antibody screen on recipient.

Crossmatch.

Retain patient specimen and unit segment at I-6°C for 7 days follouring transfusion.

416 1MMUNOHEMATOLOGY REVIEW

Crossmatches

Explanation When Performed Comnients

Antiglobulin crossmatch Recipient serum and Required when recipient donor RBCs are tested has, or previously had, a through the indirect clinically significant antiglobulin phase. antibody

Abbreviated crossmatch Recipient serum and Per~nissible if recipient Test of ABO incompatibility. An donor RBCs are tested in does not have, and has antibody screen carried through immediate spin only. never had, clinically signih- the AHG phase must be

cant antibodies performed and must he negative.

Computer crossmatch Computer check of Permissible if recipient ABO typing of recipient must be donor ABO and R h type does not have, and has done twice. Computer system and recipient ABO and never had, clinically signifi- must be validated to prevent Rh type cant antibodies release of ABO-incompatible

blood and must alert user to discrepa~lcies and incompatibil- ities.


The Major Crossmatch

Will Will Not

Detect ABO i~lcompatibility Detect all ABO typing errors

Detect most antibodies against Detect most Rh typing errors donor cells

Detect all antibodies

Detect platelet or leukocyte antibodies

Preve~it irnmullizatio~l

Ensure ~iormal survival of RBCs


The Incompatible Crossmatch

Reactions Likely Cause Resolution

One antibody screening cell and one Alloantibody Identify antibody. Crossinatch units negative donor positive in AHG. Negative for the corresponding antlgen. autocuntrol.

Antibody screening cells. all donors Positive DAT on donor Keturn unit to collecting facility. except one, and autocontrol negative at 37'C and in AHG. One donor positive in AHG only.

Antibody screening cells, donors, Lirarni autoantibody Best not to transfuse. If unavoidahle, find and autocontrol positive in AHG. "least incompatible" unit. -- - Antibody screening cells, donors. and Rouleaux Saline replacement technique autoco~itrol positive at 37'C and negative in AHG.


Emergency Transfusions Give ABO and Rh compatible if time allows for typing, otherwise, give 0 negative RBCs.

Label must indicate that crossmatch was not completed.

Physician must sign emergency release.

Routine testing must be completed and physician notified immediately of any incompatibility.


Newborn Testing*

Specimen Comments

ABO and Rh typing Cord blood, capillary, or venous blood ABO forward grouping only. Only required once per admission.

Antibody screen Serum or plasma of mother or baby Only required once per admission

Crossmatch Serum or plasma of mother or baby If the antibody screen is positive, perform an antiglobulin crossmatch on units negative for corresponding antigen.

If the antibody screen is negative, crosslnatch is not required.

'Younger than 4 months.

IMMUNOHEMATOLOGY KEVIEW 421 1

Conditions for Reissue of Blood RBCs were maintained between 1-10°C. (Red cells at room temperature reach 10°C in 313 minutes.)

Closure was not I~roken.

At least one segment remains attached to unit.

Unit is ~rlspected priol to release.

Records indicate that blood has been reissued.


Transfusion Reactions -.

5 ~ e Clinical Signs Cause Laboratory Findings Other

Hen~olytic, Fevcr; clrills; shock; Immediate destruction 111 post-transiusio~l Most serious reaction. i~~trdvascul.tr rerlal failure: DIC; of donor RBCs by specimens: hemoglobin May be fatal. Usually due

p i n ill chest, back, recipient antibody in urine and serum, to ad mini strati or^ of ABO- or flank positive n~ixed field DAT inco~npatible blood.

(unless donor cells arc all tlrstroyed), decrease ill haptoglobin, de- creasetl hernoglohin and lierrlatocrit [H and 13)

-- He~nolvtic, Fe~~er . c 3 n e ~ n i ~ , nrild Donor IiBCs are sensi- Increased bilirubirr. May he d u r to a n a ~ n ~ r e s - extr~vasrular jaundice 2 or more tized by recipient IgG positive mixed-field LIAT. tic response. Kidd allti

days after antibocly and renloved decreased haptoglobii~, bodirs most common transfusion fro111 circulation. decreased I 1 and H at 2 cause. Usuallv not liie-

or mol-e days after threatening. tr,r~lsfusion

Febrile An illcrease in Allti-leukocyte anti- None trnipcraturr 2 1 ° C bodies tvitliin 24 hours of transiusion, with rio other explanation

Common. Most often seen in multipiv tsa~is- fusrd patients or women with multiple prrgrlan- cies. Future trar~sfusions should be with leuko- poor RBCs. Antipyretics

Continrieptl

IMMUNOHEMATOLOGY KEVIEW 423

Transfusion Reaction Investigation

Specimens needed Pre-transfusion blood Post-transfusion blood Post-transfusion urine Segment from unit Blood bag with administration set and attdched IV solutions

Immediate steps Check all IDS and labels. Check post-transfusion specimen for hernolysis. Perform a DAT on post-transfusion specimen.

Signs of a he~nolytic reaction Hemolysis in post-transfusion spec i~~~er l , but not in prc-transfusion specimen. I Mixed field agglutination in DAT on post-transfusion specimen, but not on pre- I

transfusion specimen. I Further steps if ~hese are signs Check hernoglohin in first voided urine after transfusion. I of a hemolytic reaction Repeat ABO and Rh on pre- and post-transfusion specimens and unit.

Repeat antibody screen on pre- and post-transfusion specimens and unit. I

Repeat crossmatch on pre- and post-transfusion specimens. I

Additional procedures Haptoglobin (decreased with hemolysis). I Gram stain and culture of unit. I

Bilirubin 5-7 hours after transfusion (sign of extravascular hernolysis). BUN and creatinine (sign of renal involvement).


Transfusion-Associated Diseases

Infection Prevention Comments

HIV Donor screening Estimated risk: 1 in 676,000 transfusions I

Hepatitis B Donor screening Estimated risk: 1 in 66,000 transfusions. I

Hepatitis C Donor screening Estimated risk: <1 in 100,000

HTLV-I and -11 Donor screeiling Rare in United States

Syphilis Donor screening Limited risk in refrigerated or frozen components since spirochetes can't survive in cold. Highest risk from platelets.

Malaria Donor history Plasrrlodiurrz is transmitted in RBCs.

Babesiosis Donor history Babesia is transmitted in RBCs.

Chagas' disease Donor history Typanosorna crlui is transmitted in blood. Potential risk in donors

i from Central and South America.

Cytomegalovirus Selected screening CMV is transmitted in WBCs. Risk to premature infants, bone mar-

l row recipients, other iinmunocon~proinised recipients.

Graft-versus-host Irradiation of cellular Viable T lymphocytes in donor blood attack recipient. Usually disease components fatal. Blood components should be irradiated for in~munocornpro-

mised recipients, for patients with leukemia and lymphoma, and for recipients of blood from a first-degree relative, intrauterine or 1 exchange transfusion, or bone marrow transplant. a

426 IMMUNOHEMATOLOGY REVIEW I !

Hemolytic Disease of the Newborn

ABO Rh

Mothers at risk Usually group 0 Rh negative

First child affected? Yes Not usually

Frequency Common Uncommon - - -

Severity Mild Severe

DAT Weak positive or negative Strong positive

Best elution method Heat or freeze-thaw Acid or orgmic solvents

Spherocytes? Yes No

Predictable? Yes (antibody screen)

Preventable! No Yes (Rh immune globulin)

IMMUNOHEMATOLOGY REVIEW 427 1

Rh Immune Globulin (RhIG)

Composition Anti-D der~ved from pools of human plasma

Use Prevent immunization to D

Administration Antepartum: To Rh-negative woman at 28 weeks of gestation Postpartum: Within 72 hours of delivery when Rh-negative womari delivers Rh-positive

baby Other obstetric events: To Rh-negative woman after sponta~leous or therapeutic abortion,

ectopic pregnancy, anlniocentesis, chorionic villus sampling, anteparturn hemorrhage. or fetal death

Other: May also be administered to Rh-negative recipients of Rh-positive blood or components

Prenatal evaluation ABO and Rh, including weak D. If Rh positive, womari is not a candidate for RhlG. Antibody screen. I f positive, identify antibody. If anti-D is present, woman is not a

candidate for RhIG.

Postpartum evaluation ABO and Rh, including weak D, on baby. If baby is Rh negative, mother is not a candidate for RhIG.

I f baby is Rh positive, draw mother's blood within 1 hour after delivery and perform rosette test to screen for large fetal bleed. If positive, perform acid-elution test (Klei- hauer-Betke) to quantitate fetal bleed. (Fetal cells resist acid elution and stain pink. Adult cells lose hemoglobin and appear as "ghosts.")

Dose One dose per 15 mL of D-positive fetal RBCs (30 mL of fetal whole blood)

Note Residual anti-D from anteparturn RhIG may be detected following delivery and does not disqualify woman from receiving postpartum RhIG.

428 IMMUNOHEMATOLOGY REVIEW 1

Allogeneic Blood Donor Criteria (AABB)

Age At least 17

Weight At least 110 lb to donate 525 mL

Donation interval 8 weeks

Blood pressure Systolic 5180 , diastolic 5100

Pulse 50-100 with no pathological cardiac irregularities

Hemoglobin 212.5 g/dL

Temperature 537.S°C


Blood Donor Deferrals (AABB)

Deferral Condition

3 days Aspirin-containing medications i f donor is sole source of platelets

2 weeks Measles, mumps, polio, or yellow fever vaccines

4 weeks Rubella vaccine

G weeks

12 months Syphilis Gonorrhea Animal bite HBIG Tattoo Mucous membrane exposure to blood Skin penetration with sharp contaminated with blood or body fluids Household or sexual contact with individual with hepatitis Sexual contact with individual with HIV or at high risk Travel to area endemic for malaria

3 years Malaria, or from an area endemic for malaria

Contirrned

430 IMMIJNOHEMATOLOGY REVIEW

Anticoagulants/Additives/Rejuvenating Solutions

Name Abbreviation CLassification Shelf-Life Comments 1 I

Acid-citrnte-dextrose ACD Anticoagulant/ 21 days I preservative I

Citrate-phosphate- CPD Anticoagulant/ 21 days Higher pH preserves 2,3-diphospho- I dextrose preservative glycerate (2,3-DPC) better. Better 0,

delivery.

Citr~te-phosphate- CPDA-1 Anticoagulant/ 35 days Adenine incre~ses adenosine diphos- dextrose with preservative phate (ADP). adenine

Adsol AS- 1 Additive 42 days Additive is in satellite bag. After plasma is removed, additive is added to RBCs. Provides nutrients for irn- proved viability.

Nutricel AS-2 Additive 35 days Additive is in satellite bag. After plasma is rernuved, additive is added to R6Cs. Provides nutrients for im- proved viability.

Nutricel AS-3 Additive 42 days Additive is in satellite bag. After 1 plasma is removed, additive is added to RGCs. Provides nutrients for irn- proved viability. I

Contiiiued 1 IMMUNOHEMATOLOGY REVIEW 433

Donor Serological Testing Required by AABB and/or FDA

ABO

Rh (including weak D)

Antibody screen

RPR

HBsAg

Anti-HBc

Anti-HCV

Anti-HIV 1 and 2

HIV- I - Ag

Anti-HTLV-I and 11


Autologous Transfusions

Advantage No risk of disease transmission, incompatibility, or immunization

Age limit None

Hemoglobin Not less than 11 g/dL

Frequency of donation Every 3 days, but not within 72 hours of surgery

Contraindications Bacteremia

Testing ABO and Rh. If the unit will be transfused outside the collecting facility, HBsAg, HIV-1- Ag, Anti-HIV-l and 2, anti-HCV, anti-HRc, and rapid plasma reagin (RPR) are also required.

Labeling "Autologous Donor," "For Autologous Use Only." Biohazard label if any hepatitis or HIV test is confirmed to be positive.

Pretransfusion testing ABO and Rh of recipient and unit

IMMUNOHEMATOLOGY REVIEW 437 1

Blood Components

Storage Component Preparation Ten~perature Shelf-Life Indicatiorls Other

~ - ~ ~ - - ~~ - - - - - - - - - ~ ~~~ ~~~~ ~

Red blood Separated from 1-6°C 35 days in Anemia Test at least 4 units per cells (RBCs) whole blood CPDA-1,42 ~ n o n t h . 90Y1 sllc~uld

by ceutrlfugation days in AS-1 have hernatocrit of 80% or sedimentation or less. Hematocrit > any time hefore 80%: inadequate I the expiration preservative to support date of the storage. One nit whole blooct should increase hernat-

ocrit 3 % . I

RBCs froze11 Frozcn in glycerol 40% glycerol: 10 years from Anemia Check final wash for withirl 6 days of - 6 5 ° C . phlebotoniy, free hemoglobin to I , collrction 24 hours after ensure adequate glyc-

20%) glycerol: cleglyceroli- erol removal. Virtually I 5-120°C. zation all plasma, anticoagu- I

1 lant, WBCs, and i

I-6°C after de- platelets are removed. , glycerolization. Safe for IgA deficient

pdtients. Used tu store I rare cells.

- Washed RBCs RBCs washed 1-6°C 24 hours after Anelliic patients One way of producing

with saline washing with paroxysnlal leuko-reduced KBCs. nocturnal

-- I Continued

$38 IMMUNOHEMATOLOGY REVIEW

Blood Components Continued

Storage Component Preparation Temperature Shelf-Life Indications Other

whichever recipients, donor T cells occurs first recipients of blood inl~nunoincompetent.

horn a relative, bone rnarrow transplant patients

Fresh fro7en Plasnla separated plasma from whole blood

and frozen within 8 hours of collection

Cryopre- Prepared by cipitate thawing FFP

between I -G°C, removing plasma, and refreezing within 1 hour.

5-18°C. After thawing, 1-6°C.

I -18°C. After thawing, room temperature.

12 months. After thawing, t~ansfuse within 24 hor~rs.

Deficiency of coagulation factors

12 months. After thawing, tlans- fuse within 6 flours if unit is not entered, within 4 hours if pooled.

Hemophilia 11, von Willehrand's disease, Iiypo- fibrinogenemia, Factor XI11 deficiency

Check for evidence of thawir~g and refreezing. Thawed at 30-37°C or hy an FDA-approved microwave.

Check for evidence of thawing and refreezing. Thaw at 30-37" C. Test at least 4 units/month. Should contain at least 80 IU of Factor VlII and 150 rng of fibrinogen.

Contirrucd

440 1MMUNOHEMATOI.OGY REVIEW

Changes in Stored Blood

Increased Decreased

Plasma K+ PI 1

Pldsmd NH, ATP

Plasma hemog1oL)in 2,3-DPG

Mirroaggregdtes Vlable RBCs, WRCs, and platelets

J,dbile coagulat~on factors


Blood Bank Quality Control

Centrifuges Check rpm with tachometer quarterly. Check timers with stopwatch quarterly. Uetermine optinium speed and time for different procedures upon receipt, after repairs, and

semiannually.

Cell washers Check volume of saline and AHG in each tube. Verify time and speed of centrifugation periodically.

Waterbaths Check temperature daily.

Heat blocks Check temperature daily. Periodically check each well.

Refrigerators System to monitor temperaturr continuously and to record temperature at least every 4 hours. Alarm system with audible signal. Must be 1-6°C.

Freezers Syslem to monitor temperature continuously and to record temperature at least every 4 hours. Alarm system with audible signal.

Alarms Check high and low temperatures of activation quartrrly.

Platelet incubators System to monitor temperature continuously and to ~ecord temperature at least every 4 hours. Should be 20-24°C. Check rpm periodically.

Pipettes and droppers Determine average delivery volume. Calculate number of drops that will give 8O:l serum- to-cell ratio.

Continued


Changes in Urine at Room Temperature Bacteria multiply and may cause turbid~ty and a positive protein reactloll.

Bacteria convert urea to ammonia, which increases pH.

Bacteria metaholi~e glucose.

RBCs lyse in dilute or alkalilie urine.

Casts lyse in dlkaline urine.

WBCs disintegrate.

Bilirubin/urobilii1oge11 are lost through exposure to light and/or oxidation.

Ketones are lost through evaporation.

448 URINALYSIS AND BODY FLUIDS REVIEW

Urine Volume

1200-1500 mL Normal dailv vol i~me

Nor~ilal d ~ y - n i g h t ratio 211-3:l

Diuresis lncreaseci urine production

Polyuria B2000 mL/day (e.g., diabetes mellitus, diabetes insipidus)

Oligi~ria 1.500 mL/day (e.g.. dehydration, renal disease, obstructio~i of urinary tract)

Anuria No urine production - I

URINALYSIS AND BODY FI.UIUS REVIEW 449

Urine Color and Clarity

Urochrome Normal yellow color

Dilute urine Colorless 1 Concentrated urine Dark yellow, amber

Bilirubin Yellow-brown or olive-green. Yellow foam on shaking.

Homogentisic acid Normal on voiding. Brown or black on standing, beginning at surface.

Melanin Brow11 or black on standing

Methemoglobin Brown or black due to oxidation of hemoglobirl in acid urine

Myoglobin "Cola" on standing

Blood/hemoglobin Pink or red when fresh. "Col,~" or "smoky" on standing. Cloudy with RBCs. Clear with hemoglobin.

Porphyrin Port-wine

Drugs, medications, food Green, blue, orange

Pst~udomonas infection Green, blue-green

Urobilinogen Colorless when excreted. Oxidized to orange-red 01. orange-brown urobilin. i Crystals. WBCs, RBCs, Cloudy epithelial cells, bacteria

450 URINALYSIS AND BODY FLUIDS REVIEW I

Chemical Urinalysis by Reagent Strip

Test Normal Principle Significance Sources Of Error* Comments I

PH First AM: 5-6 Double buffer Useful in evalua- Decreased: Acid- Acid with proteinjmeat I Random: 4.5-8 system tion of acid-base nlnover from protein diet. Alkaline with

bala~lce, manage- square. Increased: vegetarian diet. ment of UTI and Specimen left at renal calculi room temperature

too long.

i Protein Negative-trace Protein-error Renal disease False-positive: Highly Buffered to maintain

of indicator buffered or alkaline pH 3. Most sensitive to

i i

urine, prolonged dip- albumin. Blood, WBCs, ping. False-negative: bacteria can cause posi- Proteins other than tive reaction. Orthostatic albumin proteinuria: a benign

condition in which protein is negative in the

I first AM specimen and positive after standing.

Glucose Negative Glucose oxi- Diabetes False-positive: Conta- Specific for glucose. dasejperoxi- mellitus nlination with peroxide More sensitive and dase or oxidizing detergents specific than copper

(bleach). False-negative: reduction test. For High levels of ascorbic diabetic monitoring. acid, glycolysis. specimen collected 2

Continued

Chemical Urinalysis by Reagent Strip Continued

Test Normal Principle Significance Sources Of Error* Comments

hours after eating is preferred. Normal renal threshold = 160-180 mg/dL.

Ketones Negative Sodium nitro- Increased fat Decreased in improperly Most sensitive to prusside metabolism, e.g., stored specimens acetoacetic acid reaction diabetes mellitus,

vomiting, star- vation, low carbo- hydrate diet

Blood Negative Peroxidase- Renal calculi, glo- Decreased: High levels Detects RBCs, hemoglo- like activity merular disease, of ascorbic acid, nitrites, bin, and myoglobin of hemoglobin tumors, trauma, protein, specific gravity. [muscle destruction)

pyelonephritis, Failure to mix specimen. hemolytic anemia, False-positive: Men- hemolytic trans- struation, oxidizing fusion reaction, detergents, bacterial burns, infections, peroxidase. strenuous exercise

Bilirubin Negative Diazo reaction Liver disease, False-negative: Exposure Only conjugated biliary obstruction to light, oxidation to bili- bilirubin is excreted in

verdin, hydrolysis of bili- urine. ~ - - -

'Sources of error may vary with brand of reagent strip. Refer to manufacturer's package insert.


Continued

Blood Donor Deferrals (AABB) Continued

Deferral Condition

Permanent Parenteral drug use Family history of Creutzfeldt-Jakob disease aea ted with growth hormone Viral hepatitis after 11th birthday Positive HBsAg Repeatedly reactive anti-HBc, anti-HCV, anti-HTLV, or anti-HIV High-risk behavior for HIV Sole donor to patient who developed post-transfusion hepatitis, HIV,

or HTLV Babesiosis Chagas' disease


- - -- -- - - -

Principles of Labeled Immunoassays Continued

Method I)tpe of Assay Principle

wash, substrate is added. The amount of enzyme label detected is directly proportional to the amount of antibody in the serum. Indirect ELISAs are more sensitive than direct ELISAs.

Immunoenzymo- Noncompetitive The specimen is incubated with an enzyme-labeled antibody. A solid- metric assay (IEMA) Nonisotopic phase ligand (usually on glass beads) is added and the tubes are

Heterogeneous centrifuged to remove unbound labeled antibody. The amount of labeled antibody in the supernatant is directly proportional to the concentration of the ligand in the specimen. This method is more sensitive than indirect ELISAs.

Sandwich enzyme- Noncompetitive The antigen in the specimen is sandwiched between antibody attached multiplied Nonisotopic to a solid phase and enzyme-labeled antibody. Enzymatic activity is immunoassay Heterogeneous directly proportional to the amount of antigen in the test sample.

Enzyme-multiplied Competitive The antigen in the specimen and an enzyme-labeled antigen compete immunoassay Nonisotopic for binding sites on reagent antibody. When enzyme-labeled antigen technique (EMIT) Homogeneous binds to antibody, enzyme activity is inhibited. Enzyme activity is

directly proportional to the concentration of the antigen in the specimen. This is an automated method that is frequently used for drug assays.

Continued


Antinuclear Antibodies Continued

Antibody Against Specificity Sensitivity ZFA Pattern Comments

Anti-RNP Proteins com- Present in SLE, plexed with nu- mixed connective clear RNA tissue disease and

other autoimmune diseases

Speckled

Anti-histone Histone (nucleo- Present in SLE, Present in almost Honiogeneous Diagnostic of protein that is a RA, primary all patients with drug-induced major constituent biliary cirrhosis drug-induced lupus. High levels of chromatin) lupus associated with

more active and severe forms of SLE.

Multiple ANAs can be present in a specimen. More than one pattern will be observed. Fluorescence of nucleoli is due to antibody to RNA. Seen in scleroderma and polymyositis.


- - -

HIV Testing Continued

Test Detects V p e of Test Principle Comments

Indirect Virus or antibody Confirmatory Antibody test: Serum is incu- Sensitivity and specificity immuno- bated with virally infected comparable to Western blot. fluorescence cells on a glass slide. Simple and rapid to perform. assays Fluorescein-labeled antihuman

globulin is added. Antigen test: Patient cells are fixed to a slide and incubated with HlV-specific antiserum.

P24 antigen HIV antigen Screening Anti-HIV-1 bound to a solid P24 antigen precedes antibody test support is incubated with by several weeks. Positives (HIV-1 -Ag) serum. After washing, enzyme- must be confirmed by a neu-

laheled anti-HIV-1 is tralization assay. Required on added followed by substrate. blood donors.

Polymerase Viral genome Adjunct to Viral DNA from infected cells Extremely sensitive technique. chain reaction standard is amplified, then identified Will detect infections during (PCR) testing using labeled probes. "window period." Not suitable

for routine screening.

Viral culture Virus Adjunct to Virus grown on cell culture Expensive, time-consuming, standard and hazardous. Not routinely testing performed.

Antibody tests indicate exposure to HIV.


Other Serological Tests Continued

Test Common Method Reagents Interpretation Comments

C-reactive Latex agglutination Latex particles Agglutination = Sensitive, but nonspecific, indicator protein coated with inflammation of inflammation. Originally named

anti-CRP because it was thought to be an antibody to the C polysaccharide of S. pneumoniae.

Rubella Hemagglutination Viral preparation, Titer is highest Rubella = German measles = 3-day Inhibition RBCs dilution showing measles. In-utero infections cause

no agglutination. fetal death or birth defects. Greatest A titer of 8 or risk in first month of pregnancy. IgM higher = immunity antibodies in a neonate are diagnostic to rubella. of congenital rubella.

Febrile Bacterial agglu- Salmonella 0 and Positive reaction is Screening test for fever of unknown agglutinins tination H, Brucella agglutination. origin. Weil-Felix reaction: rickettsia1

abortus, antibodies cross react with OX-2, OX- Proteus OX 19. 19, and OX-K strains of Pmteus.

Widal's test: detects antibodies in ty- phoid fever, tularemia, brucellosis. Antibodies to 0 = recent infection. Antibodies to H = past infection. Best indicator of active infection is four- fold increase in titer. Nonspecific test. Infrequently ordered today.


- --

ABO Discrepancies Continued

Anti-A Anti-B A , cells A, cells B cells 0 cells Auto Possible Cause* Resolution

4+ 2+ 0 0 4+ 0 0 Acquired B antigen Check medical history for GI problem or septicemia. Type with human anti-B acidified to pH 6.0 or with monoclonal anti-B.

4 + 4+ 2 + 0 0 2+ 0 AB with cold Perform antibody panel. alloantibody Repeat forward grouping

with B cells lacking the corresponding antigen.

*Other explanations may be possible.


-- -

-. -

Rh IIfrping Sera Continued

- - - -- - - -

High-Protein Chemically Modified Anti-D Monoclonal/ Anti-D Anti-D Pvlyclonal Blend Saline Anti-D

Comments High protein content Reacts in immediate IgM anti-D reacts with Lower incidence of enables reagent to spin without high pro- D antigen in immediate false-positives with react in immediate tein concentration. spin. IgG anti-D enables antibody coated RBCs spin. False-positive Lower incidence of reagent to be used to results if RBCs have false-positives with detect weak D. Lower a positive DAT. antibody-coated RBCs. incidence of false-positives

with antibody-coated RBCs.


- -- -- -

Frequency of Other Selected Blood Group Antigens Continued

Antigen Whites (%) Blacks (%)

Note: Students do not need to memorize frequencies but should know which antigens are common, which are uncommon, and which show marked racial differences.


- - -- . --

- -

Antibody Characteristics

Naturally occurring ABO, Lewis, P,, MN, Lua

Clinically significant ABO, Rh, Kell, Duffy, Kidd, SsU

Warm antibodies Rh, Kell, Duffy, Kidd

Cold antibodies M, N, P,

Usually only react in AHG Kell, Duffy, Kidd - - - - -

Can react in any phase of testing - ~ ~

Lewis

Detection enhanced by enzyme treatment of test cells Rh, Lewis, Kidd, P,

Not detected with enzyme treatment of test cells M, N, Duffy

Enhanced by acidification M

Show dosage Rh other than D, MNS, Duffy, Kidd

Bind complement I, Kidd, Lewis

Cause in vitro hemolysis ABO, Lewis, Kidd, Vell, and some P,

Labile in vivo and in vitro Kidd

Common cause of anamnestic response (delayed transfusion reaction) Kidd

Associated with paroxysmal nocturnal hemoglobinuria Anti-P

Continued

-- - -

Transfusion Reactions Continued

5 v e Clinical Signs Cause Laboratow Findings Other

(aspirin, acetaminophen) can be used to premed- icate.

Allergic Hives (urticaria) Foreign plasma None proteins

Common. Treat with antihistamines. Transfu- sion reaction investigation not required. Future transfusions should be with washed RBCs.

Anaphylactic Bronchospasms Anti-IgA in an IgA- None reaction deficient recipient

Rare. Transfuse with washed RBCs or blood from an IgA-deficient donor.

Circulatory Coughing, cyanosis, 'Itansfusion of too None overload difficulty breathing large a volume

Problem in children, cardiac and pulmonary patients, elderly

Septicemia Fever, cramps, Contaminated blood Positive culture on unit Can be fatal diarrhea, vomiting, muscle pain, DIC, shock, renal failure


- - -

Anticoagulants/Additives/ Rejuvenating Solutions Continued

Name Abbreviation Classification Shelf-Life Comments

Phosphate-inosine- PIPA pyruvate-adenine

Rejuvenating solution

Used to salvage rare or type 0 units up to 3 days beyond expiration. Cells are incubated with solution, then washed. (Inosine might be toxic to recipient.) Must be transfused within 24 hours or frozen.


- - - ___ _ _ - - --

Blood Bank Quality Control Continued

Antisera Test with positive and negative controls each day of use. Use heterozygous cells for positive controls.

~ - -

Reagent cells Check for hemolysis. Test daily with positive and negative controls.

Antihuman globulin Check anti-IgG activity by testing Rh-positive cells sensitized with anti-D.

QC records Retain 5 years or longer.


-

- -


Storage Component Preparation Tempemture Shelf-Life Indications Other

Platelets Centrifugation of 20-24°C 5 days with Prevent or stop Test at least 4 units/ whole blood agitation bleeding in month. 75% should maintained at patient with contain 5.5 x 101° room temperature thrombocytopenia platelets and pH 26.0. within 8 hours or abnormal One unit should raise of phlebotomy. platelet function. platelet count by Enough plasma to Contraindicated 5,000-10,00O/pL in a maintain pH at in thrombotic 75 kg recipient. 6.0 or higher. thrombocytopenic Shouldn't be sole 35 mL is mini- purpura (TTP), source of platelets mum; 50-70 mL idiopathic if donor had aspirin preferred. Can also thrombocytopenic within 3 days of dona- be prepared by purpura (ITP). tion. Should not be apheresis. used if visible aggre-

gates are present.

Granulocytes Cytapheresis 20-24°C 24 hours Neutropenia with 'Ransfuse as soon as infection possible after collection.

Must be crossmatched because RBCs are present.



Storage Component Preparation Tempemture Shelf-Life indications Other

hernoglobinuria, antibodies to IgA, or history of febrile reactions. (Deglycerolized RBCs are preferred.)

---- - - - - -- - - - - - ~ - - - - - - - - - - - - - - - - - - - - -

Leukocyte Best prepared by 1-6°C Closed system: Anemia with Must retain 80% of reduced RBCs filtration same as RBCs. history of RBCs. To prevent febrile

Open system: febrile reactions reaction, no more than 24 hours. 5 x 106 leukocytes.

Rejuvenated Treated to restore 1-b°C 24 hours after Anemia Rejuvenating solution is RBCs 2,3-DPG and ATP rejuvenation added 3 days after col-

up to 3 days after if not frozen lection to 3 days after outdate expiration. Not for use

with cells in additive solution.

RBCs, 1500-5000 rads 1-6°C Original outdate Intrauterine trans- For prevention of irradiated prior to adrninis- or 28 days from fusions, immuno- graft-versus-host

tration irradiation, compromised disease. Renders

Continued


. - - -

-


Test Normal Principle Significance Sources Of Emr* Comments

rubin diglucuronide, high levels of ascorbic acid or nitrites, drugs causing atypical colors. False- positive: Urine pigments.

Urobili- 1 mg/dL or 1 Ehrlich's Liver disease, False-positive: Porphobili- Reagent strips do not nogen Ehrlich unit reaction hemolysis nogen (with some brands detect absence of

@-dimethyl- of reagent strips) urobilinogen, only aminobenzal- increase. dehyde)

Nitrites Negative Greiss Urinary tract False-negative: Non- Test first AM reaction infection nitrate-reducing bacteria, specimen.

insufficient dietary nitrate, high levels of ascorbic acid, some antibiotics, reduction of nitrites to nitrogen, insufficient bladder incubation. False-positive: Bacterial contamination, medications that color urine red.

*Sources of error may vary with brand of reagent strip. Refer to manufacturer's package insert. Continued

URINALYSIS AND BODY FLUIDS REVIEW 453

Confirmatory/Supplemental Urine Chemistry Tests

Substance[s) Test Detected Principle Sources of Error Comments

- - ~ ~ ~ ~ - - - ~ ~ - - - - - ~~~~ ~~ ~ - - - - --

Suliosalicylic Protein Acid precipita- False-positive: Radiographic Detects all proteins, including acid tion dyes, tolbutamide, some Bence Jones proteins

antibiotics, turbid urine. False-negative: Highly buffered alkaline urine.

Clinitest Reducing Copper False-positive: High levels of substances reduction ascorbic acid. False-negative:

Glycolysis, pass through. (Color goes through orange and returns to blue or blue- green. Repeat using two-drop method and two-drop color chart.)

Nonspecific. Reacts with glucose, galactose, fructose, pen- tose, maltose, lactose. (Sucrose is not a reducing sugar.) Test all infants to diagnose galac- tosemia. Not as sensitive for glucose as reagent strip. Self- heating method. Perform in rack to avoid burning.

Acetest Ketones Sodium nitro- False-negative: Improperly Most sensitive to acetoacetic prusside reaction stored specimen acid

Ictotest Bilirubin Diazo reaction Decreased: Exposure to light, More sensitive than reagent improperly stored specimen, strip. Less affected by high levels of ascorbic acid, interfering substances. nitrites. False-positive: Urine pigments.

- - ~

Continued


Effect of High Levels of Ascorbic Acid on Urinalysis Tests

False-Positive False-Negative or Decrease*

Clinitest Glucose Blood Bilirubin Nitrite Leukocyte esterase

'May vary with brand of reagent strip. Refer to manufacturer's package insert.


- - - --- -- - --

-

Cells in the Urine Sediment

Cell Description Origin Clinical Significance Comments

Squamous 40-50 pm. Flat. Lower urethra, Usually none. Improperly epithelial cell Prominent round vagina collected clean-catch

nucleus. specimen.

Transitional 20-30 pm. Renal pelvis, ureters, Seldom significant May form syncytia epithelial cell Spherical, pear- bladder, upper

shaped, or poly- urethra hedral. Round central nucleus.

Renal tubular Slightly larger than Renal tubules nbular necrosis, toxins, Differentiate from polys by epithelial cell a WBC. Round. viral infections, renal adding 2% acetic acid to

Eccentric round rejection visualize nucleus nucleus.

Oval fat body Renal tubular Renal tubules Same as renal tubular Maltese crosses with polar- epithelial cell con- epithelial cells ized light taining fat droplets

White blood Usually polymor- Kidney, bladder, or Cystitis, pyelonephritis, 0-5/high-power field (HPF) cell (WBC) phonuclear. Approxi- urethra tumors, renal calculi are normal. Clumps of

WBCs are associated mately 12 pm. with acute infection. Granular appearance.

Continued

458 URINALYSIS AND BODY FLUIDS REVIEW - -- -

m e Description Significance Comments

Hyaline Homogenous with parallel 0-2/low-power field Most common type. Least significant. sides and rounded ends (LPF) are normal. Contain Tamm-Horsfall protein only. Dissolve

Increased with stress, in alkaline urine. Same refractive index as fever, trauma, exercise, urine, so may be overlooked if light is too renal disease. bright.

Granular Same as hyaline, but 0-l/LPF is normal. May originate from disintegration of cellular contain granules Increased with stress, casts

exercise, glomerulonephritis, pyelonephritis.

RBC RBCs in cast matrix. Acute glomerulonephritis, Pinpoints source of bleeding in kidney. Most Yellowish to orange color. strenuous exercise fragile of casts. Often in fragments.

Blood Contain hemoglobin. Same as RBC cast From disintegration of RBC casts Yellowish to orange color.

WBC Leukocytes incorporated Pyelonephritis into cast matrix. Irregular in shape.

Pinpoints kidneys as the site of infection

Epithelial Renal tubular epithelial cells Renal tubular damage Transitional and squamous epithelial cell cell incorporated into cast matrix casts do not exist. These cells are found dis-

tal to renal tubules and collecting ducts where casts are formed.

Continued


- -

Crystals Found in Acid or Neutral Urine

Crystal Description Significance Comments

Amorphous urates Irregular granules None Form pink precipitate in bottom of tube. May obscure significant sediment. Dissolved by warming to 60°C.

Uric acid Very pleomorphic. Four- Usually normal Birefringent. Polarizes light. sided, six-sided, star- shaped, rosettes, spears, plates. Colorless, red- brown, or yellow.

Calcium oxalate Octahedral (eight-sided) Normal. From Occasionally found in slightly alkaline urine. envelope form is most oxalate-rich Monohydrate form may be mistaken for RBCs. common. Also dumbbell foods. Most common constituent of renal calculi. and ovoid forms.

Leucine Yellow, oily-looking Severe liver Often accompanied by tyrosine spheres with radial and disease concentric striations

Qrosine Fine yellow needles in Severe liver Often accompanied by leucine sheaves or rosettes disease

Cystine Hexagonal (six-sided) Cystinuria Must be differentiated from uric acid. Does not polarize light. Confirm by cyanide-nitro- prusside test.

Continued


- - --- - -

- -- - - - --

Crystals Found in Alkaline Urine

Crystal Description Significance Comments

Amorphous phosphates Irregular granules None Form white precipitate in bottom of tube. Dissolve with 2% acetic acid.

Triple phosphate "Coffin-lid" crystal None

Ammonium biurate Yellow-brown "thorn None Seen in old specimens apples" and spheres

Calcium phosphate Needles, rosettes, None Only needle form seen in alkaline urine "pointing finger"

- - - - - - - - - - - - - - -

Calcium carbonate Colorless dumbbells or aggregates None


-

- - - -

Miscellaneous Urine Sediment

Structure Description Significance Cornrnen ts

Bacteria Rods, cocci Urinary tract infection or Usually accompanied by WBCs contaminant when clinically significant, unless

patient is neutropenic

Yeast 5-7 pm. Ovoid, colorless, Usually due to vaginal or fecal Differentiate from RBCs by adding smooth and refractile. May contamination. May be due to 2% acetic acid. RBCs are lysed; be budding. kidney infection. May be seen yeast are not. Presence of pseudo-

in urine of diabetic patients. hyphae indicates kidney infection.

Sperm 4-6 pm head with Usually not significant in an 40-60 pm tail adult. May be a sign of sexual

abuse in a child. - - -

Trichomonas Resembles WBC. Rapid, Contaminant from genital tract Should not be reported unless jerky, nondirectional motility. infection motile

Mucus Transparent, long, thin, Large amount seen with chronic May be mistaken for hyaline casts ribbonlike structure with inflammation of urethra or tapering ends bladder


L- -- -- -

Renal Disorders

Disorder Cause Reagent Strip Sediment Other

Acute glomer- Inflammation and Protein, blood RBCs (some dysmorphic), Frequently follows a ulonephritis damage to glomeruli WBCs, RBCs, and/or group A strep infection

hemoglobin casts

Nephrotic Increased glomerular Protein (large Casts (all kinds), free fat Hypoproteinemia, syndrome permeability amount) and oval fat bodies hyperlipidemia

Pyelonephritis Infection of upper Protein, leukocyte WBCs, WBC casts, urinary tract involving esterase, nitrite bacteria interstitial tissue of kidney

Cystitis Bladder infection Leukocyte esterase, WBCs, bacteria, possibly nitrite RBCs. No casts.


- - - -

Cerebrospinal Fluid

Normal Abnormalities Comments

Color Colorless Xanthochromia = slight pink, Examine for xanthochromia within 1 hour orange, or yellow supernatant. of collection to avoid false-positives due to Due to oxyhemoglobin or bili- lysis of RBCs. Centrifuge specimen and rubin. Seen with subarachnoid compare to water. hemorrhage. Red or pink, uniformly distributed in all tubes = subarachnoid hemorrhage. Red or pink, diminishing from tube 1 through 3 = blood from traumatic tap.

Clarity Clear Cloudy with infection or bleeding

WBC Adult: 0-5 lymphs/pL. Increased in meningitis Perform cell count in a hemacytometer Newborn: 0-30 within 30 minutes of collection

monos/pL.

RBC 0 Increased with subarachnoid Uniform distribution with subarachnoid hemorrhage or traumatic tap hemorrhage. Uneven distribution with

traumatic tap. (More in tube 1.)

Glucose 60-70% of blood Decreased in bacterial Blood glucose method glucose meningitis

Continued


- -- -- - -- - - - --

Meningitis

Bacterial Viral Mycobacterial Fungal

WBC Increased Increased Increased Increased

Differential Polys Lymphs Lymphs Lymphs

Protein Increased Increased Increased Increased

Glucose Decreased Normal Decreased Normal or decreased

Lactate Increased Normal Increased Increased

Other Weblike clot or pellicle


- - - - - - -- - -

Body Fluids

Definition Other Terms

Effusion Abnormal accumulation of fluid in a body cavity. Classified as transudate and exudate.

Serous fluid Fluid contained in pericardial, peritoneal, and pleural cavities

Pericardial fluid Fluid surrounding heart Pericardiocentesis fluid

Peritoneal fluid Fluid in abdominal cavity Abdominal fluid, ascitic fluid

Pleural fluid Fluid surrounding lungs Chest fluid, thoracentesis fluid, empyema fluid

Synovial fluid Fluid in joints Joint fluid


- --

Transudates and Exudates

Ransudate Exudate

Etiology Systemic disorder that affects fluid Condition involving membranes within filtration and reabsorption; e.g., con- the body cavity; e.g., infection, malig- gestive heart failure, hypoalbuminemia, nancy, inflammation, hemorrhage. cirrhosis. Problem originating outside of the body cavity.

v p e of process Noninflammatory Inflammatory

Color Colorless Yellow, brown, red, green

Clarity Clear Cloudy

Specific gravity (1.015 >1.015

Protein t 3 g/dL >3 g/dL

Fluidserum protein t 0 . 5 >0.5

Spontaneous clotting No Yes

LD (lactate dehydrogenase) (200 U/L >ZOO U/L

F1uid:serum LD <O.G >0.6

WBC < 1 ,OOO/pL >l,OOO/~L

Differential Predominantly mononuclears Predominantly polys


- -- - - - - - - -- - -


Test Normal Principle Significance Sources Of Error* Comments

Leuko- Negative Granulocytic Urinary tract False-positive: Oxidizing Will detect intact and esterase infection agents. Decreased reaction: lysed polys.

esterase reaction High glucose, protein, Lymphs do not specific gravity, or react. ascorbic acid.

Specific Random PKa change of Indication of kid- Increased: Protein. Measures ionizable gravity specimen: polyelectrolyte ney's concentrat- Decreased: Alkaline urine. substances only. Not

1.003-1.030 ing ability and always the same as state of hydration specific gravity by re-

fractometer.

'Sources of error may vary with brand of reagent strip. Refer to manufacturer's package insert.


- -

Cells in Body Fluids

Fluid - -

Normal Cells Abnormal Cells

Cerebrospinal fluid Lymphocytes RBCs Monocytes Neutrophils Ependymal cells Eosinophils Choroid plexus cells Basophils

Siderophages Blasts/malignant cells (Nucleated RBCs and cartilage cells may be present from tap)

Serous fluid (peritoneal, Lymphocytes RBCs pericardial, pleural) Monocytes/histiocytes Neutrophils

Mesothelial cells Eosinophils Basophils Lupus erythematosus (LE) cells Malignant cells

Synovial fluid Lymphs RBCs Monocytes/histiocytes Neutrophils Synovial cells LE cells

RA cells (ragocytes) Malignant cells (rare)


-- - -- - - - - -

Confirmatory/Supplemental Urine Chemistry Tests Continued

Substance(s) =st Detected Principle Sources o f E m r Comments

Watson- Schwartz Test

Urobilinogen, Ehrlich's alde- Decreased: Exposure to light, porphobilinogen hyde reaction more than 1 hour at room

temperature. False-positive: Warm aldehyde reaction. (Urine should be at room temperature.)

Hoesch Test Porphobilinogen Ehrlich's alde- Similar to Watson-Schwartz hyde reaction

Collect specimen from 2-4 PM. Store in dark. Urobilinogen is soluble in chloroform and butanol. Porphobilinogen is not soluble in either.

Urobilinogen doesn't react unless very high.


Body Fluid Cells

Cell Significance Size Cytoplasm Nucleus Comments

Mesothelial Normal in serous Variable. Abundant. fluids. From lining 12-20 pm Basophilic. of pericardial, May have pleural, and peri- vacuoles toneal cavities. and frayed

Synovial Normal in syno- borders. vial fluid. From synovial membrane.

Oval. About one-half cell diameter. May be eccentric. Dark purple. Stippled. May have nucleoli and peri- nuclear clear zone. May be multinu- cleated (similar size and shape).

"Fried egg" appearance. May be in singles, pairs, or sheets. Homogenous cells of uniform size. Each retains its individuality. Slitlike opening (window) between cells.

Histiocyte Found in normal 15-30 pm Abundant. Round or irregular. Abnormal forms: (Macrophage) fluids Colorless. Eccentric. Delicate erythrophage (contains

May chromatin. RBCs), siderophage have many (contains siderotic vacuoles granules), signet ring and ingested (large lipid vacuole matter. that pushes nucleus

to the edge)

Malignant Wmor Great Scant Often multiple with Bizarre appearance. cells variation variation in size. Irregular in shape.

Hyperchromatism. Tendency to fuse. Nucleoli. May have Cells lose individuality. mitotic figures. No window between

cells. Nuclear molding. 3D appearance.


Cells in the Urine Sediment Continued

Cell Description Origin Clinical Significance Comments

Glitter cell WBC with Brownian Same as WBC Same as WBC Seen in hypotonic urine movement of granules. Stain faintly or not at all.

Red blood Biconcave disk, Kidney, bladder, Infection, trauma, Crenated in hypertonic cell (RBC) approximately 7 km. or urethra tumors, renal calculi. urine. Lyse in hypotonic

Smooth. Non- Dysmorphic RBCs urine and with 2 % nucleated. indicate glomerular acetic acid.

bleeding.

URINALYSlS AND BODY FLUIDS REVIEW 459

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