Clinical chemistry SEH

Define isoelectric point

The pH at which an amino acid/protein has no net charge

Describe protein metabolism

Proteins in the body repetitively synthesized (anabolism) & degraded (catabolism) allowing the recycling of amino acids

How are proteins synthesized?

• Transcription/Translation

List protein functions

• Enzymes

• Transport carriers

• Receptors

• Structure & support for cells

• Antibodies

• Hormones

• Acid-base balance

What are the two classifications of proteins?

• Simple

• Conjugated/complex

Total protein is made up of 2 proteins/groups, what are they?

• Albumin

• Globulins

What does the term acute phase reactant/APR mean?

Decreases in the presence of acute disease states, such as inflammation, infection, or injury

What is the main clinical significance of prealbumin?

It migrates before albumin in classic serum protein electrophoresis. Transport protein for thyroid proteins

What are the functions of albumin?

• 80% of colloid osmotic pressyre

• Ability to bind and transport a large variety of substances throughout the body

What is the major alpha 1-globulin?

Alpha-1 Antitrypsin (AAT)

What is AFP used to screen in expecting mothers?

Fetal abnormalities and stress at 15-20 weeks

A decrease in haptoglobin is seen in which medical conditions?

• Hemolytic anemia

• Cirrhosis

Ceruloplasmin contains what trace element in plasma?

Copper

What is the name of the rare autosomal recessive trait that the ceruloplasmin is low and the copper concentrations are increased?

Wilson’s disease

What protein is the major component of β-globulins and the principal plasma protein for transport of iron?

Transferrin

Which protein is an acute phase reactant and a nonspecific indicator of bacterial or viral infection, inflammations and tissue injury or necrosis

C-Reactive Protein (CRP)

What are the five classes of immunoglobulins?

• IgG

• IgA

• IgM

• IgD

• IgE

What are some causes of hyperproteinemia

• Result of dehydration

• Excessive production of proteins

What are some causes of hypoproteinemia

• Decreased synthesis

• Increased protein catabolism

• Low intake of proteins and amino acids

What is the most common protein methodology for protein?

Total protein

Name two other methodologies used for protein testing

• Dye binding

• UV absorption

What is the reference range for protein

6-8.3g/dL

What are the two common dyes used for albumin methodology

• Bromocresol green

• Bromocresol purple

What is the reference range for albumin

3.5-5.5 g/dL

What is protein electrophoresis?

Migration of proteins based on their density & charge under the influence of an electric field

What types of specimens can be used for a protein electrophoresis

• Serum

• Urine

• CSF

A normal electrophoresis has how many bands? Name the bands

• 5 bands; Albumin, Alpha-1, Alpha-2, Beta, Gamma

What kind of bands are looked for in CSF for a MS patient? Are these bands present in a SPEP?

• Oligoclonal bands

• They are not present

What type of proteins are monitored for active demyelination in MS patients

Myelin Basic Protein

What type of protein might be found in a urine electrophoresis of a patient diagnosed multiple myeloma?

Bence-Jones proteins

If plasma is used, what kind of band show up in the beta-gamma region?

A distinct fibrinogen band

What abnormalities would you see on the protein electrophoresis with the following medical conditions?

Cirrhosis – Beta-Gamma bridge

Nephrotic syndrome- Decreased albumin, Alpha1-globulin, Beta globulin, gamma globulin, Increased Alpha-2 globulin

Multiple myeloma- M-spike in the gamma region

alpha 1-antitrypsin deficiency- Increase in everything but the Alpha-1 globulin

What follow up test would be ordered if there was a monoclonal gammopathy

Immunoelectrophoresis to confirm immunoglobulin and/or light chain

What proteins are tested on an immunofixation electrophoresis?

specific types of immunoglobulins (antibodies) and their protein building blocks (heavy and light chains)

What is myoglobin?

The primary oxygen-carrying protein found in striated skeletal and cardiac muscle. 2% of total muscle protein

What color will urine be if myoglobin is present?

Reddish-brown

What is the prosthetic group in hemoglobin and myoglobin?

Heme group

What are porphyrias?

A group of rare inherited or acquired metabolic disorders caused by loss or gain of function mutations in the enzymes responsible for heme synthesis

How are porphyrias categorized?

Erythropooietic or hepatic

What is urea?

The major excretory product of protein metabolism is amino acids. and is present in highest concentration in the blood

The amount of urea absorbed back into the blood stream depends on what two things?

• Urine flow

• Extent of hydration

The concentration of urea in plasma is determined by what four things?

• Dietary protein intake

• Protein catabolism

• GFR

• Hydration status

What is the measurement of urea used for?

Used to evaluate renal function, address hydration status

What is the most common method used to measure urea?

Couples the urease reaction with glutamate dehydrogenase

Define the following: azotemia, uremia

• Azotemia is elevated concentration of uria in the blood

• Uremia is a very high urea level in the blood accompanied by kidney failure

What is the normal BUN/creatinine ratio

10:1 to 20:1

What is gout and how is it diagnosed?

a painful type of inflammatory arthritis caused by a buildup of sharp uric acid crystals in the joints, and it is diagnosed using a uric acid test

What is the name of the enzyme that humans and higher primates don’t have in their bodies to further breakdown insoluble uric acid to the soluble metabolite, allantoin

Uricase

What other medical conditions would uric acid be tested for?

• Gout

• Uric acid nephropathy

• Purine metabolism

• Kidney dysfunction

• Renal calculi

What is the normal range for uric acid in men and women?

Male: 3.5-7.2 mg/dL

Female: 2.6-6 mg/dL

What chemical component forms creatinine?

Creatine

Name the three amino acids known to synthesize creatine in the liver

• Arginine

• Glycine

• Methionine

What serves to be a high energy source for muscles?

ATP

Plasma creatinine concentration is a function of what three things?

• kidney damage progression

• kidney disease

• kidney function

What is creatinine clearance? How is it calculated

• A measure of the amount of creatinine eliminated from the blood by the kidneys

• Urine creatinine * Urine flow rate/Plasma Creatinine=mL/min

What is the calculation for a 24 hour urine creatinine level?

Urine Creatinine/100* Total Urine Volume

Name the two reasons laboratories were encouraged to report an estimated GFR

• Improve patient care

• When serum creatinine is ordered as a means to increased identification of kidney disease

What are the four variables used in the current MDRD equation for eGFR

• Serum creatinine concentration

• Age

• Gender

• Race

What is the method frequently used to measure creatinine

Jaffe Reaction

What type of specimens are acceptable to analyze urea, uric acid and creatinine

• serum

• plasma

• urine

What is ammonia?

Produced in the deamination of amino acids during protein metabolism

What medical conditions are ammonia levels considered useful?

• Hepatic failure

• Reye’s syndrome

• inherited deficiencies of urea cycle enzymes

What method is the most common technique used to measure ammonia?

GLDH

Name the two anticoagulants that are suitable to use for collecting an ammonia level.

• Heparin

• EDTA

How should specimens for ammonia levels be handled?

placed on ice immediately after collection and separated from cells within 15 to 30 minutes

What should patients refrain from for several hours before having an ammonia level collected?

Smoking, strenous exercise, anything other than water drank

How is water used in clinical chemistry?

Preparation of reagents

• Diluent for controls and calibrators

• Flush/clean internal components of analyzers

• Serve as a medium in heating bath to incubate cuvettes

• Wash/rinse laboratory glassware

What are the six different classifications of water by CLSI standards?

1. Clinical Laboratory Reagent Water (CLRW)

2. Special Reagent Water (SRW)

3. Instrument fee water

4. Water supplied by method manufacturer

5. Autoclave and wash water

6. Commercially bottled purified water

Which classification of water is acceptable to use with reagents, controls and calibrators?

Clinical Laboratory Reagent Water (CLRW) or Type 1 Reagent Grade

What is a primary standard? How is it used in clinical chemistry?

A standard is a solution with a known concentration of an analyte.

• It is used as a calibrator to set the correct value when performing a calibration

Describe what “calibration” means?

The calibration of an instrument is the process that pairs an analytical signal with a concentration value of analyte. It is used to establish a relationship between the concentration of the analyte and the magnitude of the optical or potentiometric signal given by the measuring device

When is it necessary to perform a calibration?

1. New reagent lot number

2. QC results do not meet the acceptable limits

3. After maintenance of critical parts/subsystems or after service procedures are performed

When should quality control be performed?

1. Every 24 hours or each shift, according to laboratory policy

2. After calibration has been performed

3. Questionable patient results

What is a calibration verification (linearity study)?

A process of testing materials of a known concentration in the same manner as patient samples to assure the test system is accurately measuring samples throughout the analytical measurement range

If reagents are kept refrigerated, what is the acceptable temperature range? Room Temperature?

• 2°-8°C

• 15°-30°C

Name the procedure that must be performed to make a new lot number of reagents ready for patient use

Reagent Lot Number Verification

What types of glass pipettes are “TD”? “TC”?

• TD “To Deliver” - Volumetric

• TC “To Contain” - Serological (Measuring/Graduated)

o Requires “blow out”

Name the 3 types of automatic pipettes used in the laboratory?

1. Fixed-volume

2. Adjustable-volume

3. Multichannel

What is considered a micropipette? Macropipette?

• Micropipette is a pipette that dispenses less than 1 mL.

• Macropipette is a pipette that dispenses more than 1 mL

List the 2 ways that pipettes can be calibrated?

1. Gravimetric method (Most desired method)

2. Photometric method

Describe the characteristics of “low actinic glass”.

Low actinic glass has a high thermal resistance with an amber color to protect the contents from light and visualization of the contents.

Name the different types of volumetric glassware.

• Erlenmeyer/Griffin

• Graduated cylinder

What are the 3 variables in centrifugal force

1. Mass – 1.118 x 10-5

2. Speed – RPM

3. Radius (r) – Distance from the center of rotation to the bottom of the tube in the rotor cavity/bucket during centrifugation

How is RCF calculated?

RCF = 1.118 X 10-5 x r x (rpm)²

List the 5 types of rotor heads that can be used in a centrifuge

1. Fixed angle

2. Angled (Vertical)

3. Swing bucket

4. Cytocentrifuge

5. Hematocrit

Which is the most common rotor head used in clinical chemistry?

Swing bucket

Describe the following rotors – swing, fixed angle and vertical

• Swing – Buckets swing out into horizontal position, allow better separation of individual particle types.

• Fixed angle – Tubes held fixed at an angle of the rotor cavity, most used for “pelleting”.

• Angled (Vertical) – Tubes held in vertical position during rotation

List the types of specimens that can be used in clinical chemistry

• Serum

• Plasma

• Whole blood

• Urine

• CSF

• Other body fluids

Which specimen type is preferred for STAT testing

Plasma (Specimen drawn in a lithium heparin tube

Define lipemic, hemolysis and icteric.

• Lipemic – Increased amounts of triglycerides/fats in a patient sample that make the serum/plasma appear “milky”.

• Hemolysis – Destruction of red blood cells that causes the serum/plasma appear pink to red.

• Icteric – Increased amounts of bilirubin that causes the serum/plasma to appear yellow to yellowish green.

Define photometry

the science of measuring visible light in terms of how the human eye perceives its brightness

What is the wavelength range that the lab commonly uses in testing?

190 nm to 1100 nm

What is Beer’s Law?

The concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light

Be able to describe the relationship that absorbance and the % transmittance has with a sample concentration.

Negative exponential relationship

List the 5 components of the spectrophotometry.

• Light source

• Monochromators

• Sample Cell

• Photodetectors

• Readout Display

What type of light sources are used for UV and visible wavelengths

Visible—incandescnet tungsten or tungsten-iodide lamp

UV— deuterium lamp and mercury arc lamp

What is the monochromator used for in the spectrophotometry

Isolates individual wavelengths of light

What types of monochromators are there that can be used

• Colored glass

• Interference filters

• Glass prisms

• Diffraction gratings

What are the 4 types of a photodetector

• Photocell

• Phototube

• Photomultiplier

• Photodetectors

What type of material is used for a cuvet if it is to be read in the UV range

Quartz