special chem and electrophoresis

study questions

describe the principle of testing protein electrophoresis on the Helena SPIFE

proteins are large molecules composed of covalently linked amino acids. Depending on electron distributions resulting from covalent or ionic bonding of structural subgroups, proteins can be either polar or nonpolar at a given pH. they are separated according to their respective electrical charges on an agarose gel and then stained to visualize the distribution of the proteins

what is the difference between protein electrophoresis and immunofixation electrophoresis on the Helena

electrophoresis-divides proteins based on size and charge

immunofixation- used to ID monoclonal immunoglobulins; antiserum is used; heavy and light chains can be ID

what is the first peak seen in a normal gel serum electrophoresis pattern

albumin

what is the second peak seen in a normal gel serum electrophoresis pattern

alpha 1

what is the third peak seen in a normal gel serum electrophoresis pattern

alpha 2

what is the fourth peak seen in a normal gel serum electrophoresis pattern

beta (technically beta 1 and 2 as both peaks are included under one category)

what is the last peak seen in a normal gel serum electrophoresis pattern

gamma; anything after the beta peak is considered gamma

which charge is by the albumin side of the gel serum electrophoresis pattern

cathode (pos)

which charge is the cathode

positive

which charge is the anode

negative

which charge is near the gamma end of the gel serum electrophoresis pattern

anode (neg)

which directions of migration do proteins take in the gel serum electrophoresis

anode to cathode

what is the normal range for total serum protein

6.3-8.2 g/dL

what happens when hemolyzed serum is used for serum protein electrophoresis

increased alpha-2 and beta peaks due to elevated hemoglobin and haptoglobin

what happens when plasma is used instead of serum for gel serum protein electrophoresis

fibrinogen will appear as an extra band between beta and gamma

is plasma or serum used for serum gel electrophoresis

serum!

what fraction does haptoglobin migrate to

alpha 2

what fraction does C3 migrate to

beta 2

which fraction does IgG migrate to

Gamma

what fraction does alpha-1-antitrypsin migrate to

alpha 1

what fraction does transferrin migrate to

beta 1

what does a electrophoresis pattern with nephrotic syndrome look like (which peaks are affected?)

an increase in alpha 2 and beta peaks; decreased albumin

what does a electrophoresis pattern with cirrhosis look like (which peaks are affected?)

increase gamma to create a beta-gamma bridge; decreased albumin

what does a electrophoresis pattern with multiple myeloma look like (which peaks are affected?)

increased gamma peak; monoclonal gamma should also be increased

what does a electrophoresis pattern with hypogammaglobulinemia look like (which peaks are affected?)

gamma is decreased

which protein fraction would be influenced by severe nutritional deficiency

decrease in albumin

which protein fraction would be influenced by iron deficient anemia

beta 1 increased

which protein fraction would be influenced by nephrotic syndrome

alpha 2 and beta would increase; albumin would decrease

which protein fraction would be influenced by acute inflammatory disorders

alpha 1, alpha 2, beta 1 and beta 2 would increase

which protein fraction would be influenced by immunosuppression due to chemotherapy

decreased gamma

briefly list the steps in the procedure for immunofixation electrophoresisi (IFE or serum)

1) specimen applied to 6 lanes

2) electrophoresis performed

3) Ab applied to matching lane

4) Ab attaches to Ag present , forming complex

5) membrane is stained, destained and then dried

6) visual interpretation

what is the purpose of the immunofixation electrophoresis (IFE urine or serum)

ID heavy and light chain immunoglobulins

are the gels used in electrophoresis acidic or alkaline

alkaline

define gammopathy

primary disease state in which a single clone of plasma cells produces elevated lvls of an immunoglobulin of a single class or type

state the normal range for a 24 hr urine protein

42-255 mg

explain the sample prep necessary before electrophoresing a urine sample

1) allow sample to come to room temp

2) mix well, pour into a blue aliquot tube for protein quantititation on track and into a clear 12 mL centrifuge tube

3) spin tube for 10 min

4) pipete prepped smaple into concentrator tube, fill to top, spin 15-18 min at 3300 RPM or until the lvl of concentrated sample is at 250 uL

define bence jones proteins

monocolonal free light chains produced in excess by plama cells and found in urine

what is the significance of bence jones proteins in urine

highly suggestive of neoplastic process and is often associated with a greater severity of the underlying disease

what may be included in multiple myeloma treatment

stem cell transplant, chemotherapy, Ab therapy, immunomodulators, protease inhibitors

state the physiological functions of hemoglobin in the body

transports O2 from lungs to tissues; transports CO2 waste back to lungs

what is the range for Hb A1 in a normal adult

95-98%

what is the range for Hb A2 in a normal adult

1.5-3%

what is the normal amount of Hb F present in an adult

0-2% (trace)

what are the hemoglobins normally found in a healthy adult

Hb A1, Hb A2, Hb F

explain the significance of elevated Hb A2 lvls

could signify carriers of beta thalassemia

what are the hereditary causes of elevated Hb F lvls

hereditary persistence of Hb F, Sickle Cell

what are the acquired causes of elevated Hb F

pernicious anemia, leukemia, PNH, pregnancy, cancer metastatic to the bone, hyperthyroidism, chronic kidney disease

what does PNH stand for

paroxysmal nocturnal hemoglobinuria

what does PNH do

premature destruction of RBC

by what age is Hb F normally replaced

6 mo

define the term ‘Hemoglobin structural variant’ and list some examples

-qualitative globin abnormalities, genetic mutations substitute amino acids in one of the globin chains; sickle and alpha/beta thalassemia

define thalassemia

genetic mutations result in decreased synth of one or both of the globin chains (alpha and beta)

what are the types of thalassemia

alpha thalassemia, major/minor thalaseemia

describe the clinical presentation of beta thalassemia

major is lethal, severe anemia, jaundice, splenomegaly, bone malformations, stunted growth

sickle cell disease demonstrates what symptoms and lab results

chronic hemolytic anemia, pain crisis, vascular occlusion, organ damage, increased susceptibility to infections; elevated Hb F

describe the test principle used by the Capi 3

capillary electrophoresis; charged molecules are separated by their electrophoretic mobility in an alkaline buffer with a specific pH (9.4), separation occurs according to the electrolyte pH and electroosmotic flow

what testing does the Phadia do

allergy testing

describe the sandwich immunoassay principle involved in ImmunoCap allergy testing

1) specific IgE in pt serum reacts w/ the immunocap which is saturated with the target antigen

2) the cap is then washed and Anti-IgE labeled with an enzyme is added to form a complex. after incubation, unbound enzyme-labeled anti-IgE is washed away

3) a developing solution is added (substrate) and incubated

4) the rxn is stopped with stop reagent and the fluorescence of the product of the enzyme rxn is read in the fluorometer

how is fluorescence related to the Ab present in the sample

the fluorescence is directly related to the amt of allergen-specific Ab present in pt sample

what are the two major phase of immediate hypersensitivity

Sensitization and then activation

what is sensitization in hypersensitivity

IgE is formed after exposure to allergen. The IgE binds to effector cells at the Fc region

what is activation in hypersensitivity

cell-bound IgE binds to the allergen and triggers degranulation of the effector cell it’s bound to. Inflammatory meidators such as histamines and prostaglandins are released

what is another name for immediate hypersensitivity

Type 1 hypersensitivity

what does the aglient test for

lead

briefly describe the principle of electrothermal atomic absorption spectrometry (graphite furnace) used in the blood lead procedure

1) pt spec diluted 1:10 with matrix modifier, which places lead in a constant form and minimizes matrix interferences

2) spec is charred in a graphite tube to remove interfering substances, then atomized. the lead vapor absorbs light at 263.3 nm wavelength

3) a lead hollow-cathode lamp (HCL) shines at 283.3 nm light through the vapor

how does light absorbed related to amt of lead present

the amt of light absorbed is directly related tot he amount of lead present in a sample

list common sources of lead exposure

paint chips, contaminated soil, smelting, mining, ammunitions, soldering, plumbing, ceramic glazing

what kind of sample is used for lead testing

whole blood

why are clotted specimens rejected

can cause false decrease in results

what are the affects of lead poisoning in children

headaches, seizures, coma, GI symptoms, acute nephropathy, anemia, severe cognitive and behavioral problems

what are the affects of lead poisoning in adults

peripheral neuropathies, motor weakness, chronic renal insufficiency, systolic hypertension, anemia

what are the affects of low level lead exposure

symptoms increase as lead accumulates and may not be symptomatic until a certain threshold is reached

what does the optilite measure

free light chains in urine as well as cystatin C lvls

describe the tubidimetric principle as it apples to the optilite

the amt of light transmitted is indirectly proportional to the concentration of the analyte in the test sample

more analyte → more turbid → less light shines through

how does the optilite id samples that are in antigen excess

activity check- rate of rxn at different time points is assessed to ID samples that had a relatively high int’l concentration. if detected, will auto dilute samples

what does the activity check look like for samples with excess Ag

they will show a high int’l rate of rxn that will rapidly slow as Ab becomes saturated

what does the activity check look like with a non excessive Ag samples

steady rxn rate

list some of the diseases associated with elevated serum lvls of monoclonal free light chains

multiple myeloma, lymphocyte neoplasma, Waldenstrom’s Macroglobulinemia, AL amyloidosis, light chain deposition disease, connective tissue diseases like SLE

when would Cystatin C testing be indicated

confirmation for Dx of kidney disease; a decreased GFR

how does Cystatin C related to GFR

as GFR decreases, Cystatin C increases; Cystatin C is produced by all nucleated cells at a constant rate and freely filtered by golmerulus and almost completely reabsorbed and degraded by proximal tubular cells

what is GFR based on

serum creatinine

what does the Indiko test for

anti-convulsants

what are the two drugs specifically tested for using the Indiko

lamotrigine and topiramate

what method is used on the Indiko plus

tubidimetric immunoassay