Week 11 Flashcards
3 major classes of plasma proteins
albumin, globulins and fibrinogen
Function of albumin
low levels are seen in
testing usually done with
maintains Collodial osmotic pressure
regulates fluid balance
most abundant protein in blood
liver disease, nephrotic syndrome, malnutrition, or chronic inflammation
tested with bromocresol green or purple DYE BINDING METHOD - calibrate and use controls- avoid hemolysis
Function of Fibrinogen
protein in blood clotting
converts to fibrin to form clot
HIGH levels in ACUTE inflammation, pregnancy, and birth control use
LOW levels in liver disease, DIC, and malnutrition
how do you know if you have nephrotic syndrome
swelling because you have low protein = low plasma proteins
albumin is lost through urine
proteinuria- frothy urine - DIPSTICK
hypoalbuminemia - muscle wasting - tiredness HALLMARK
hyperlipidemia - lipid in urine
edema (periorbital swelling and anasarca
glomerulus injury -proteins leak into urine
renal biopsy
macroscopic testing detects lipid casts and oval fat bodies
how to do you know you have liver disease
altered albumin and globulin levels will show you have -cirrhosis- alchohol, Hep B/C, NAFLD
-hepatitis-inflamed liver due to virus A-E
-fatty liver disease- insulin resistance, AFLD from alcohol
-Liver cancer - hepatocellular carcinoma HCC - primary liver cancer
symptoms
juandice
ascites - fluid in abdominal cavity
hepatomegatly
coagulopathy
What will you see in Chronic Inflammatory and Autoimmune Disorders
-increased gamma globulins because your immune system is constantly stimulated
-protein electrophoresis can help to differentiate between polyclonal and monoclonal
RA, SLE and IBD
increase of cytokines TNF alpha
autoantibody production
What quality things can affect plasma protein testing
Sample quality- hemolysis, lipemic and icteric samples
Calibration and controls - regular calibration and QC with EVERY BATCH
check to make sure there are no interferences like medications or paraproteins
What is CRP
initatites opsonization and encourages phagocytosis
-marker of early phase reactions and increases with inflammation, bacterial and viral infections, MI, RA, gout
increased when there is liver disease, malnutrition and protein losiing
risk factor for CVD, inflammation and atherosclerosis
-part of non specific response
-increases quickly in 6-12 hours of inflammation and does down once the inflammation goes down
-flareups associated with IBD or SLE
hs CRP = increased with atherosclerosis, coronary artery disease, and atherosclerosis
monitored post op to detect infections
IgG associated with
autoimmune disease and hepatic disease
IgA associated with
skin, gut, respiratory infections, cirrhosis
found in secretions like tears
IgM associated with
viral infections, blood infections (malaria), and acute hepatitis.
IgD associated with
surface receptor for antigens on B lymphocytes
primary function unknown
IgE associated with
histamine release from mast cells
asthma, allergies and parasites
what are poly and monoclonal increases in gammaglobulins associated with
decreases in gammaglobulins associated with
poly increases = chronic infections, liver disease, autoimmune and parasite infections
mono increases - myeloma , Waldenström
macroglobulinemia
decreases = leaves you vulnerable to diseases and can order through primary or secondary causes like sickness or drugs
What is the albumin/globulin ratio
-gives you albumin ratio by dividing up the globulin concentration
-RI is 1.0-1.8 variations can occur because of lab methods
-looks at liver function, immune function
Globulins = TP - ALB
then A/G ratio is A/G
What does a low A/G ratio tell you
less than 1.0
-increased globulins and decreased albumin
CHRONIC liver disease - reduced protein syn
Nephrotic syndrome - albumin lost through urine because of kidney damage
Chronic inflammatory or autoimmune disorders- RA, Lupus = high globulins
Myeloma, plasma cell dyscrasias - over production of gammaglobulins
what does a high A/G ratio tell you
high than 2.5
Hypogammaglobulinemia- Common Variable Immunodeficiency - CVID
what does increased vs decreased Total protein tell you
myeloma, chronic infections, and dehydration.
decreased:
nephrotic syndrome, severe burns, liver disease,
inflammation, and malnutrition.
how are protein levels measured
Spectrophotometry, immunoassay, nephelometry, electrophoresis, and mass spectrometry
What is the Biruet method of measuring total protein
color change that occurs when cupric ions are in alkaline solution
-color intensity is proportional to protein concentration looked at through spectrophotometer
-hemolysis, icterus , lipemia can cause issues with measurement
-contains
CuSO4, NaOH, NaK tartrate, KI.
How is albumin measured
bye binding
Nephelometry, turbidimetry for specific proteins.
What do abnormal SPE show you
patterns for nephrotic syndrome and acute phase proteins.
Abnormal SPE in Various Conditions
how is liver cirrhosis seen on SPE
Increased β-γ globulins indicative of cirrhosis.
how is Hypogammaglobulinemia seen on SPE
Decreased or absent γ-globulins, suggesting immune deficiencies.
how is Monoclonal Gammopathy seen on SPE
M-spike in γ-globulin region, common in multiple myeloma., Waldenström’s macroglobulinemia, and MGUS
What other testing indications will you find in multiple myeloma
bence jones protein = kappa or lamba free light chains
increased IgG, hypercalcemia, renal insufficiency, anemia and bone lesions
What is Waldenstrom Macroglobulinemia
non-Hodgkin’s lymphoma
severe fatigue , bleeding and anemia
can use IFE to detect = IgM monoclonal proteins
how does IFE work and what are its problems
two stage procedure
separate proteins according to motility and immunoprecipitated to detect abnormal Immunoglobulins
used for monoclonal proteins looking for MS
if there is AG excess , artifacts, improper washing it can cause inaccuracies in the IFE results
How can refractometery be used to measure proteins
uses the refractive index of serum which changes with the protein concentration in serum
-less accurate/specific than biuret method
-is affected by glucose and electrolytes in sample
What is an interference in the dye binding method
high amounts of globulins, bilirubin = over estimation of albumin levels
What is the immunoassay method for albumin detection
use antibodies specific to albumin to detect and
quantify its concentration.
AG-AB complexes are measured using nephelometry, turbidimetry
more costly
What is SPE
separates and quantifies proteins in serum based on size, shape and charge
proteins separate into distinct bands: albumin, alpha-1, alpha-2, beta, and gamma globulins.
What is the gel in Agarose gel electrophoresis stained with
protein-specific dye (e.g., Coomassie Brilliant Blue), and a densitometer is used to quantify the bands corresponding to different protein fractions (albumin, alpha-1, alpha-2, beta, and gamma).
a broad increase in gamma region of electrohoresis gel tells you
its a poly clonal increases
interferences in agarose gel electrophoresis
Hemolysis (free hemoglobin), lipemia (high levels of lipids), and paraprotein precipitation can cause artifacts that affect the interpretation of results.
What is Capillary Electrophoresis
serum proteins are seperated in capillary tubes based on mobility in an electric field
-faster and better resolution
-uses an electrophoretogram to quantify protein fractions and detect monoclonal bands
What is immunofixation electrophoresis IFE
detects and characterizes monoclonal proteins (also known as M-proteins or paraproteins) in serum,
urine, or cerebrospinal fluid (CSF).
used after SPE when a monoclonal protein is detected
-used to differentiate hematologic malignancies
What is the gold standard for testing for multiple myloma
IFE
determines the heavy and light chain
What is MGUS
Monoclonal Gammopathy of Undetermined Significance
detected by IFE
looking for monoclonal proteins in asymp proteins to monitor progression of multiple myeloma
What is the procedure for IFE
first stage is like SPE where the proteins separate into distinct bands: albumin, alpha-1, alpha-2, beta, and
gamma globulins on gel with electric current
then antisera is applied to different gel sections to detect immunoglobulin heavy chains (IgG, IgA, IgM, IgD, IgE) and light chains (kappa and lambda).
-antisera binds to target proteins and forms immune complexes = fixed on get
-gel is then washed to removed anything that is unbound
-gel is stained with protein specific dyes like Coomassie blue or Amido Black , destained to remove excess dye
look at bands under imaging system
what are common concerns for IFE troubleshooting
sample handling - make sure there is not hemolyis, lipemia or contamination
faint/absent bands - due to insufficient concentration improper antisera- REPEAT TEST WITH HIGHER SAMPLE CONCENTRATION
non specific binding - due to excessive background, wash carefully
distorted or diffuse bands - uneven polymerization , sample overloading
how is CRP measured
immunoturbidimetry - changes in turbidity when CRP binds to ANTI crp antibodies
-measured with spectrophotometer and calculated based on calibration curve
-lipemia, hemolysis or icterus can impact test
how is High-Sensitivity CRP (hs-CRP) Testing done
Immunonephelometry:
measuring light scatter by CRP AB complexes = detects low levels of CRP which is necessary for CVD
-concentration related to Calibration curve
-sensitive can detect down to 0.1mg/L = good for detection of low grade inflammation
hemolysis and lipemia can affect
<1 = low cardiovascular risk
>3 high risk
How can ELISA be used to measure CRP
-binding CRP to surface bound AB followed by a 2ndary AB linked to enzyme , then a substrate which that is changed by the enzyme to a detectable signal
has a microplate coated with anti CRP antibodies
good for use in research
-but very labor intensive
how can POCT be used for CRP testing
lateral flow immunoassay
capillary or venous
easy to use
less precise than lab test
hemolysis, lipemia and icterus is an issue
-different methods are best for specificity levels
What tests would you order if you think someone had liver disease
ALT/AST - increased = hepatocellular injury
ALT = liver specific
AST = heart and muscles
ALP/GGT - increased in cholestasis and bile duct obstruction
Bilirubin - increased = liver dysfunction
ALB =decreased
PT - prolonged
Viruses - check for hepatitis
ANA, ASMA, Anti LKM
Imaging - Ultrasound, CT and MRI
What tests to order if you think someone has Chronic Inflammatory and Autoimmune Disorders
Autoantibody tests: RF and ACPA = RA
ANA = SLE
anti ds DNA and Anti SM - very specific for SLE
ANCA= vasculitides or granulotomas
inflammatory markers - CRP, ESR
CBC
MRI and ultrasound for RA and arthritis
endoscopy for IBD
