HCMC Chemistry Critical Competencies

List the types of pipettes and pipetting devices used

• Volumetric (transfer)

• Mohr (measuring)

• Serological (measuring)

• Micropipettes/mechanical

• Bulb

• Pipette pump

• Propipette

Describe the volumetric pipette

• Designed to deliver a single volume with high accuracy.

• Consists of a cylindrical bulb joined at both ends to narrow glass tubing.

• Colored bands on the top of the pipette denote the volume that is dispensed. These
  are used when reconstituting QC and calibrators

• Above the bulb is a calibration mark (ring). Fluid must be drawn up the pipette to above the ring and then released slowly until the bottom of the meniscus is exactly at the ring. To transfer the volume, touch the pipette tip to the inside of the container and allow liquid to drain out (do not blow the remaining drop out).

• Several sizes (1,2 and 5 mL).

Describe Mohr (measuring) pipettes

• these pipettes are graduated but stop at a baseline before the pipette begins to narrow (tip).

• To accurately transfer fluid, the meniscus must be
  precisely on a mark both at the beginning and end of transfer.

Describe serological (measuring) pipettes

• Graduated so that multiple volumes can be delivered.

• Typical volume range from 0.2 mL to 10mL and have graduation marks that continue
  to the tip. These are blown out.

• Calibrated TD (to deliver) with etched rings at the top.

• Several sizes: 5, 10, 25.

Describe micropipettes/mechanical

• can be set to drawn and dispense different volumes.
  This is done by turning the knob at the top. The tips are disposable.

• To draw liquid up, depress the plunger to the first stop and immerse the tip into the liquid. Release
  the plunger back to the rest position. Depress the plunger to the first stop and then press the plunger to the second stop to expel all the liquid
  

Describe the pipette bulb

• Hand squeeze the bulb and touch the opening to the mouth of the pipette. Insert into the liquid and slowly release pressure.

• Liquid will be drawn up into the pipette, and pull the bottom of the meniscus up past
  the desired level.

• Quickly remove the bulb as you slip your free index finger over the
  top of the pipette.

• Lift out of the solution and twist until desired level.

• Remove finger and allow the pipette to drain.

• Touch tip to the inside of the container after all liquid has been drained.

Describe the pipette pump

insert pump to end of the pipette.

Put the tip into the solution and turn the wheel with your thumb to draw up liquid.

Press on the wheel gently to hold the liquid in the pipette until you are ready to expel it.

To expel the fluid, turn the wheel in the opposite direction.

Touch the tip of the pipette to the flask to drain the last bit of liquid

Describe propipette

Use your thumb and forefinger to press on valve "A" and squeeze the bulb with other fingers to produce a vacuum for aspiration.

Release valve "A" once the bulb is completely deflated.

Hold the pipette close to its upper end and insert into the bottom of the
propipette.

Insert the pipette into the liquid to be transferred. Press on valve "S". Suction will draw liquid up into the pipette.

Continue pressing valve "S" until the liquid reaches the desired level.

Carefully adjust the fluid level so that the bottom of the meniscus coincides
with the calibration line on the pipette.

Press on valve "E" to expel liquid

Reading micropipettes and volumes

never rotate the volume adjustment knob past the upper or lower range of the pipetman

look at the front of the pipette and you will see a window with digits inside.

Examples:

P1000 reads 040 = 400 uL

P20 reads 125 = 12.5 uL

P100 or 200 reads 100 or 200 = 100 uL or 200 uL

P20 ranges from 2-20 uL

P200 ranges from 20-200 uL

P1000 ranges from 200-1000 uL (1 mL)

Pre-analytical variable and sample requirements

Dilutions

Define glycogen

polysaccharide that is major storage form of glucose in liver and muscles

What is the function of glycogen

• main function is to store chemical energy in the liver and muscles for later use.

• Glycogen is converted from glucose for storage and broken down into glucose when energy is needed.

How does the body regulate glycogen

• Regulated through the formation of glycogen from glucose (glycogenesis) stimulated by insulin and the breakdown of glycogen into glucose (glycogenolysis) stimulated by glucagon.

• Glycogenesis = Glucose + insulin = glycogen

• Glycogenolysis = Glycogen + glucagon = Glucose

Define glucagon

• A hormone produced by alpha-cells of islets of Langerhans of pancreas

What is the function of glucagon

• Increases blood glucose (hyperglycemic agent) and stimulates the liver to release stored glycogen as glucose.

• Helps maintain plasma glucose levels by increasing them when they are low and prevents insulin from decreasing levels too low.

• Increases blood glucose levels by stimulating the release of glycogen

How does the body regulate glucagon

• Regulated through levels of blood glucose.

• When blood glucose is low, increased levels are seen

• When blood glucose is high, decreased levels are seen

Define glucose

• Monosaccharide (simple sugar) utilized as the body’s main source of energy

What is the function of glucose

• Functions to provide energy for metabolic processes

How does the body regulate glucose

• Regulated through hormones such as insulin (decreases levels) and glucagon (increases levels).

Define insulin

• Pancreatic enzyme secreted by beta cells of the islets of Langerhans

What is the function of insulin

• Controls metabolism of sugars and is principal hormone responsible for decreasing blood glucose

• Lowers glucose levels when they are elevated

How does the body regulate insulin

• Regulated through blood glucose levels

• When blood glucose is increased, increased levels are seen

• When blood glucose is decreased, decreased levels are seen

Lipid structure

• A class of extremely hydrophobic organic compounds.

• Made primarily out of carbon, hydrogen, and oxygen.

• Classified into five classes

  • sterol derivatives

  • fatty acids

  • glycerol esters

  • sphingosine derivatives

  • terpenes.

Define HDL

• the smallest and most dense lipoprotein that make up 15-20% of total cholesterol.

• “good cholesterol”

What is the function of HDL

• Functions to return cholesterol to the liver for extraction

Define LDL

• Class of lipoproteins that are principal carriers of cholesterol, accounting for approximately 70% of total cholesterol in plasma

• “Bad cholesterol”

What is the function of LDL

• Function to transport cholesterol in the plasma to peripheral tissues

Define VLDL

• Class of lipoproteins that are primarily transporters of endogenous triglycerides from liver to muscles and adipose cells

• contain greater lipid-to-protein ratio and are least dense of lipoproteins.

What is the function of VLDL

• Functions to transport endogenous triglycerides from the liver to muscle and adipose tissue

Define cholesterol

• Sterol that contains 27 carbon atoms and four fused rings (A, B, C, and D) called a perhydrocyclopentanophenathrene nucleus

• The primary sterol derivative from lipids

What is the function of cholesterol

• serves as a structural component of cell membranes and a precursor for bile acids, vitamin D, and steroid hormones (cortisol, aldosterone, progesterone, estrogen, and testosterone).

Define chylomicrons

• largest and least dense (<0.94) of lipoprotein classes; formed from lipids absorbed in intestines

What is the function of chylomicrons

• Transports exogenous triglycerides from the intestines to the liver and peripheral cells

Define triglycerides

• the most common glycerol esters in plasma, comprising glycerol and three fatty acids

What is the function of triglycerides

• Functions as the main storage of energy in the body

• main storage of energy in the bod

Lipid digestion and absorption

Lipid- fatty acid catabolism and anabolism

lipid- cholesterol and metabolism

lipids- lipoproteins

Define acid-base balance

• the maintenance of homeostasis of the hydrogen-ion concentration of body fluids

Describe buffer systems of the blood

• First line of defense to a deviation in homeostasis due to the resistance of pH changes.

• The blood buffers included in the body are

  • bicarbonate

  • hemoglobin

  • phosphate

  • protein.

• Bicarbonate is the most important buffer system due to the large amount of CO2 and the acid potential of the body

Describe the roles of the respiratory system and kidney’s in acid-base balance

Respiratory system:

• Eliminates CO2

  • moves air in and out (external convection)

  • Consists of the lungs, airway, and respiratory muscle that maximize gas exchange

  • maintains high external PO2 and low external PCO2

Circulatory system

• internal convection system

  • Delivers blood with a low PO2 and high PCO2

Kidneys

• Maintains pH through excretion

  • Exchanges Na+ and H+

  • Produces and excretes ammonia

  • reclamation of HCO3-

Describe factors contributing to acidosis and alkalosis

• Development is due to an imbalance of acid-base concentrations.

  Acidosis:

• Factors that influence acidosis include…

  • the accumulation of acids or a loss of extracellular buffers.

  • This can be caused by conditions, such as severe diarrhea, abdominal fistulas, renal failure, or diabetes mellitus.

  Alkalosis

• results from an accumulation of alkalis or a reduction of acids.

  • This can be caused by many factors, such as vomiting, hypocalcemia, bicarbonate retention, or diuretics.

Define ALT

• An intracellular enzyme involved in amino acid metabolism

• catalyzes the transfer of an amino group from alanine to α-oxoglutarate

• present in high concentrations in the liver and one of the analytes measured in a liver function profile

What disease states is ALT elevated?

• Elevated levels are seen in…

  • hepatitis C

  • alcoholic hepatitis

  • choledocholithiasis

  • cirrhosis

Define ALP

• Enzyme with an optimal pH of 9.0–10.0

• frees inorganic phosphate from an organic phosphate monoester

• present in highest concentration in the cells of the biliary tract and osteoblasts, so elevated levels are present in obstructive liver disease and various bone disorders

What disease states is ALP elevated?

• Elevated levels are seen in…

  • primary sclerosing cholangitis

  • primary biliary cirrhosis

  • hepatitis

Define AST

• One of a group of enzymes that catalyze interconversion of amino acids and α-oxoacids by transfer of amino groups

• catalyzes deamination of aspartate to oxalacetate

• highest levels found in…

  • liver

  • muscle

  • brain

• Its measurement is included in liver function profile

What disease states is AST elevated?

• Elevated levels are seen in…  

  • Alcoholic hepatitis

  • choledocholithiasis

  • primary sclerosing cholangitis

  • NAFLD.

Define amylase

• Class of enzymes that catalyze the hydrolysis of starch

• two main sources of amylase are the pancreas and salivary glands, so serum levels are elevated in pancreatic and salivary gland disorders

What disease states is amylase elevated?

• Elevated levels are seen in…

  • acute pancreatitis

  • chronic pancreatitis

Define GGT

• Membrane-associated enzyme that transfers γ-glutamyl group from glutathione and other γ-glutamyl peptides to amino acids or small peptides to form γ-glutamyl amino acids and cysteinyl-glycine

What disease states is GGT elevated?

• elevated in various conditions involving hepatic damage, especially hepatobiliary, and may also indicate alcohol or drug toxicity.

• Elevated levels are seen in…

  • primary biliary cirrhosis

  • hepatitis

  • NAFLD.

Define direct bilirubin

• bilirubin conjugated by UDP-glucoronyl transferase that adds two glucuronic acid molecules

• water soluble and excreted in urine

What disease states is direct bilirubin elevated elevated?

• Elevated levels are seen in…

  • hepatitis

  • cirrhosis.

Define BUN and its functions

• nitrogen found in urea as distinguished from nitrogen in other blood proteins.

• Functions as a measurement for renal function

What disorders are increased or decreased levels of BUN seen?

• Elevated levels of BUN are seen in…

  • dehydration

  • congestive heart failure

  • acute glomerulonephritis

  • acute renal failure

  • nephrotic syndrome

  • acute kidney injury

• Decreased levels are seen with…

  • severe liver disease

  • overhydration

  • pregnancy

  • SIADH

Define and describe the function of creatinine

• product formed by breakdown of creatine phosphate

• functions as a measurement to assess renal function

What disorders are increased or decreased levels of creatinine seen?

• Elevated creatinine levels are because of prerenal, renal, and postrenal causes.

• Elevation is seen in…

  • acute glomerulonephritis

  • acute renal failure

• Decreased levels are seen with…

  • low muscle mass

Define and describe the function of microalbumin

• excretion of minute amounts of albumin in urine, too small to be detected by reagent dipsticks

• Functions as a measurement used to detect early onset renal disease

What disorders are increased or decreased levels of microalbumin seen?

• Increased microalbumin levels are seen in…

  • diabetic nephropathy.

Define and describe ammonia

• product of amino acid and protein catabolism that is converted to urea in liver by Krebs–Henselheit urea cycle

• functions to assess renal function

What disorders are increased or decreased levels of ammonia seen?

• Ammonia is elevated in cases of…

  • impending hepatic coma

  • advanced liver disease

  • acute viral hepatitis

  • severe gastrointestinal bleeding

  • chronic hepatitis

  • Reye’s syndrome.

Define and describe the function of potassium

• A primary cation electrolyte found in the body

• functions as a measurement for tubular reabsorption status

What disorders are increased or decreased levels of potassium seen?

• Elevated potassium levels are seen in…

  • pseudohyperkalemia

  • leukocytosis

  • vigorous arm exercise

  • metabolic acidosis.