Laboratory Essentials for Research Nurses

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Laboratory Essentials for Research Nurses

• Karen Hopcia, MS, RN-C, COHN-S

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Objectives

• Understand the importance of laboratory skills in
  clinical data collection and integrity
• Increase knowledge of specific types of data
  points
• Increase knowledge of specific sampling and
  laboratory skills for use on the GCRC

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Contents

• Types of data points
• Common laboratory equipment
• Blood sampling
• Urine Sampling

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Hierarchy of Data Collection

• Qualitative Data
• Data which is interpreted in nominal (yes or no)
  answers
• Typical of safety data and certain stimulation
  tests
• Certainty of data point around cut-off value
• Quantitative Data
• Data points which have numerical values and can
  be compared to other data points for meaningful
  evaluation
• Typical for data collection on the GCRC
• Certainty of absolute data value

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Common Data Points

• Single data points
• Multiple data points
• Pharmacokinetic studies
• Physiologic studies
• Stimulations tests

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Single Data Points

• Single data points are those in which a single
  datapoint is used as the outcome
• Examples include
• 24 hour urine Calcium or Urine Free Cortisol
• Urine Hcg
• A bone density
• CBC to evaluate WBC count before preceding with a
  study

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Multiple Data PointsPharmacokinetic (PK) studies

• Typically used to evaluate uptake,
  bioavailability and elimination of drugs from the
  body.
• PK studies can be used to compare differences
  between groups of individuals or to check drug
  interactions.

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Multiple Data PointsPhysiologic studies

• Time periods may vary from
• Minutes
• Hours
• Days
• Months
• Years
• Precision of data collection dependent on
• Amplitude of the pulse (variation from baseline)
• Frequency of the pulse (how often the pulse
  occurs)

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Multiple Data PointsStimulation Tests

• These tests are typically done to compare levels
  post administration of a stimulus to the pre-dose
  level
• The post dose points might be single points
  (qualitative test) or multiple points
  (quantitative test). Examples include
• Oral glucose tolerance test
• Cosyntropin test for adrenal function

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Common Laboratory Equipment

• Centrifuge
• Balance
• Pipetman

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Centrifuge

• Centrifuges separate blood components by using
  centrifugal force generated from spinning
• Centrifuges are available to spin a variety of
  tubes and speeds
• The GCRC centrifuges are low speed laboratory
  centrifuges that can spin up to 4000rpm
• Ultracentrifuges which are utilized to isolate
  DNA can run at speeds of up to 100,000rpm

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Rotors

• Types
• Fixed angle rotor
• Swinging bucket rotor - the bottom of the buckets
  swing outward during the spin
• Inserts
• A variety of inserts can be purchased for the
  type of tube to be spun. The tube should fit
  fairly snugly to the insert

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RCF or g force and RPM

• Relative Centrifugal Force (RCF)
• The force during centrifugation that moves the
  particles outward from the center of rotation
• Also know as the g force
• Revolutions per minute (rpm)
• The number of times the a rotor completes a
  revolution in one minute of centrifugation
• The RCF and rpm are related but dependent on the
  characteristics of the rotor (the radius)

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The radius of the rotor

• For the same revolutions per minute, the amount
  of force measured increases as the radius of the
  centrifuge increases
• Swinging bucket rotors add more to the radius as
  the bucket swings outward during the spin and
  have more force than fixed angle buckets

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Counterweight

• The centrifugal force must be equal on both sides
  of the rotor for the centrifuge to run
  efficiently, a blank is inserted to the side with
  less weight (think of a washing machine
• The higher the spin speed, the more accuracy that
  is required between the sample and the blank
  weight
• Blanks can be made by adding or subtracting water
  until the blank weighs the same as the sample
• We spin at relatively slow speeds. While we must
  include blanks for unbalanced loads, tubes within
  a cc (1gram) of the sample weight suffices for
  our purposes.

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Calculating g force from rpm

• All you need is the radius (r) from the rotor.
• The radius for both rotors is 18.54 cm.
• RCF 11.17 x (r) x(rpm ? 1000)
• To convert 3000 to g force
• RCF 11.17 x 18.54 x (2800 ? 1000) 1864

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Calculating rpm from g force

• We more commonly want to convert a force
  specified by the investigator to rpm.
• RPM ( RCF x 1000) ? (11.17 x (r))
• To convert 3100g force to RPM
• RPM ( RCF x 1000) ? (11.17 x 18.54) 2000
• Note that there is an automatic calculator on the
  X drive at X\GCRC_NU\Rotor conversion table.xls

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Balance

• There are many types of balances available on the
  market but all use digital or mechanical
  counterweights to accurately measure loads
• The tare is an amount subtracted from the final
  weight
• The tare is valuable to subtract the weight of a
  container
• Balances must be on level surfaces
• Many balances have levels (bubbles) to stabilize
  the balance
• Balances should be in areas that are free of air
  current
• Air currents can add forces which can be measured
  as weight (think about pressing your hand on a
  balance and the weight that is added

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Specific Gravity or Density

• For clinical purposes, Specific Gravity (SG) is
  used interchangeable with Density
• Density is measured as mass per unit volume
• Density Mass ? Volume or for our purposes
• Volume Mass ? Density
• Conveniently, water has a density of 1. This fact
  allows us to measure urine using a scale

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Specific Gravity of unine

• Example
• If a urine sample has a specific gravity of 1.010
  and a mass of 1000g (as measured on the scale),
  what is the volume of the sample
• Volume Mass ? Density 1000g ? 1.010g/cc
  990cc
• As you can see, since the density of urine is
  very close to water, we can substitute the weight
  for volume
• The error obtained by weighing the sample is
  990/1000 or 1 and is far less than would be
  obtained by measuring the volume using graduated
  cylinders

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Pipetman

• Pipetman are mechanical pipets used to accurately
  measure additives or aliquots
• They should only be used where precision of
  measurement is needed
• They come in various sizes including 20 ul, 200ul
  and 1ml sizes
• Care must be taken so that liquid is not
  suctioned into the reusable barrel of the pipet
• Important There are two stops to a pipetman
• The first stop is the accurate measurement of
  desired amount specified on the dial
• The second stop (using increased force) denotes
  the entire amount specified on the pipet or the
  total volume

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Preanalytic Variables

• Variables of data collection, processing and
  transport can affect the final data
  interpretation. These include
• Venipuncture technique
• Temperature and time of storage
• Handling of sample during transport to lab

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Specimen Collection

• Blood
• Sterile technique is employed for patient safety
  and to decrease the chance that bacteria will
  grow in the specimen
• Urine
• Clean technique is employed. Most bacteria find
  the acid environment hostile and growth is slow
• CSF
• Is treated similar to blood samples and can be
  spun for cells or other components
• The pellet is small and almost invisible to the
  eye

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Blood collection Hemolysis

• Hemolysis is the degradation of the cells in the
  sample. Hemoglobin produces the red pigment seen
  in the spun sample
• Hemolysis is caused by excessive force during the
  collection or spin process
• Force during collection is created by
• Excessive force generated by the
• phlebotomist
• Bends in the collection apparatus

Increased force here
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Blood Collection

• Typical laboratory assays need 100 microliters
  (0.1cc) or less
• For Red Top Tubes, the amount of blood collected
  per tube can very based on the number of tests
  and needs
• For tubes with additives, it is usually necessary
  to collect the entire quantity specified for the
  tube to maintain the blood additive ratio.

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Blood Collection Components

• On the GCRC, several blood components are of
  interest and can be categorized as
• Proteins
• Enzymes such as insulin
• Carbohydrates
• Glucose (we dont collect a lot of carbohydrates
• Metals or other salts
• Calcium that can be involved in metabolic
  processes or byproducts
• Lipids
• Hormones

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Blood Processing Centrifugation

• Blood separates with the heaviest portions
  setting at the bottom and the lightest components
  at the top
• Typically spun at 1000 to 2000g
• The faster the spin speed and the longer the
  spin, the more compact the heavy components
  become on the bottom
• Spinning too fast will cause cell lysis
• The top liquid portion of the spun tube is called
  the supernatent
• The bottom solid layer is the pellet

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Blood processing

• The percentage of red cells is directly
  proportional to the Hematocrit of the sample
• Samples with 40 red cells have a Hct of 40
• Samples that have few red cells may be subject to
  dilution during the sampling (unless anemia or
  some other cause is diagnosed)

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Blood Processing

• Handling of specimens is based on how fragile is
  the component to be measured
• Proteins and carbohydrates are typically very
  fragile
• Metals and salts are subject to alteration by
  metabolism
• Lipids such as steriod hormones are stable
• Refrigeration of samples retards proteases and
  DNAses in the blood from degrading components of
  interest, typically protein and DNA
• Disturbed (mixed)specimens should be respun
• Never aliquot red cells into an tube where
  hemolysis has not occurred (cells contain lysing
  components)

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Blood collection tubes

• Tubes are collected in a specific order based on
  perishibility
• Investigator preference for tube collection takes
  precedence over a preset order
• Serum is collected from tubes in from tubes
  without anticoagulant is not added
• Plasma is collected in tubes with anticoagulant.
  Plasma contains clotting factors

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Blood ProcessingRed Top Tube (RTT)
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Blood ProcessingSerum Separator or Red Tiger
(SST)
Note Gel tubes are a typical barrier to protect
the blood component of interest from metabolic
degradation
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Blood ProcessingGreen Top Tube
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Blood ProcessingGreen Gel or Green Tiger
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Blood ProcessingYellow Tiger Top Tube
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Blood ProcessingGray Top Tube
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Blood ProcessingNavy Top Tube
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Blood ProcessingPurple Top Tube
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Blood ProcessingLight Blue Top Tube
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Blood ProcessingYellow ACD Tube
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Aliquot tubes Materials

• Polystyrene
• Clear tubes made of plastic
• Usually the least expensive and versatile of
  tubes
• Does not freeze as well as polypropylene (can
  crack in -70 degree freezers)
• Some proteins may adhere to plastic making these
  tubes unreliable for some aliquots
• Polypropylene
• Cloudy tubes made of plastic
• Versatile tube made which can withstand higher
  spin speeds and lower temperatures than
  polystyrene

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Aliquot tubes Types

• Falcon or other aliquot tubes
• Tubes manufactured for the storage and transfer
  of specimens
• They are not sterile
• Aliquot tubes are typically inexpensive
• Cryovials
• 1 to 5cc sterile polypropylene tubes with
  sealable o-ring gaskets.
• Used for cryogenic purposes to freeze live cells
  in liquid nitrogen
• Expensive - usually 1 or more per tube
• Centrifuge tube
• Sterile 15cc or 50cc tubes used to store urines
  or large amounts of fluid
• Made to withstand the speeds of centrifuges
• Expensive

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Aliquot technique

• Depress pipet
• While controlling the force of the pipet, tilt
  the tube and start suctioning liquid from the top
  of the tube, lowering the pipet as the meniscus
  of the liquid is lowered
• Avoid the buffy coat unless specifically ordered

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Urine collection

• Collection based on times
• Single voids
• Qualitative values of urine components
• Urine HcG (highest in first morning void)
• Urine Toxicology screen
• Safety voids for protein, glucose, blood WBC etc.
  
• Timed voids, most commonly 24 hour urines
• Types of collections
• Plain
• Acid (contains 25-40cc of 6M Hydrochloric Acid
  which can cause chemical burns)
• Split 50/50 acid and plain

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Measuring Urines

• Accuracy of urine measurement is vital to split
  urines
• The amount of urine components excreted varies at
  different times of the day
• The error rate can be substantial in voids not
  collected equally
• If the volume in a split urine is different in
  the plain and urine collection by more than
  100cc, contact the investigator and document on
  the lab requisition and in your note

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What is a meniscus?

• The meniscus is the concave shape of the top of
  the liquid seen due to the surface tension of the
  liquid measured
• Measure liquids at the lowest part of the meniscus

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Use wider or taller cups?

• A taller cup has more space between the
  gradations than wide cups and approximation of
  the measurement is better
• If you have a choice between cups, a taller cup
  is preferable since it will be more accurate

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Urine ProcessingPlain Urine
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Urine Processing Acid Urine