Proteins & Tumor Markers Flashcards
1
Q
Electrophoresis
A
- Movement of charged molecules/particles in a liquid medium under the influence of an electrical field
- Used to separate proteins, mainly serum*, CSF, urine and hemoglobins
- Sample applied at cathodic (-) end of alkaline gel and voltage applied –> negatively charged proteins migrate based on net charge/size
- Proteins stained and visualized, quantified by densitometer
2
Q
Electrophoresis Media
A
- Most common is agarose gel –> polysaccharide from seaweed, neutral so separation based on mainly charge, low endosmosis
- Polyacrylamide gel –> used in IEF & nucleic acid separation, not charged = no endosmosis, particles separated by size (crosslinking fibers), medium has neurotoxic monomers (issue if made in-house)
3
Q
Electroendosmosis
A
- Support has fixed negative charges (OH-) with adjacent immobile (+) ions –> Stern Potential
- When voltage applied, solvent flows towards cathode
- Mobile ions (Zeta Potential) with low to no-charge migrate with solvent toward cathode, strong negative charges ions remain fixed to medium (or migrate slowly)
- Migration flow dependent on distance from fixed charges (less flow as farther away)
4
Q
Hemoglobin Electrophoresis
A
- Run on both alkaline and acid gels to separate Hgb variants
ALKALINE: A / F / S,D,G / C,E,O,A2
ACID: F / A,D,G,O,A2,E / S / C
- separation also done by HPLC or capillary electrophoresis
5
Q
Capillary Electrophoresis
A
- Process run in long capillary tube with each end in inlet/outlet buffer; spec (lamp&detector) set up to detect fractions
- Good heat dissipation allows for very high voltage
- Rapid separation, no staining or densitometry, highly automated
- Very high endosmosis
6
Q
Isoelectric Focusing (IEF)
A
- Ampholyte mixture buffer creates linear pH gradient along gel
- Proteins migrate to isoelectric point (pI) = pH at which protein has no net charge
- Refocusing of proteins that try to diffuse (due to gradient they are sent back to pI) allows for sharp bands
- Used for CSF oligoclonal banding (MS)
7
Q
Western Blot
A
- not used much clinically
- proteins separated by MW in gel electrophoresis
- transferred to nitro-cellulose membrane “blotting”
- Sample Ab’s bind to immobilized target proteins, washed and stain using labeled Ab
8
Q
Albumin
A
- Only protein quantified by ep, others need nephelometry/turbidimetry
- Most abundant protein in plasma (60%), maintains Colloid Osmotic Pressure (COP) and carrier for many molecules
- Hyperalbuminemia: not significant other than dehydration
- Hypoalbuminemia: increased loss (NEPHROTIC SYNDROME, gi, burns), decreased production (liver failure, malnutrition)
- Analbuminemia (little to no albumin) and bisalbuminemia (two peaks, dimeric protein)
- Being bound to bili/drugs may alter mobility
9
Q
Pre-Albumin
A
- Not a form or precursor of albumin
- Migrates before albumin on gel (seen in CSF gels, not always in serum gels)
- aka Thyroxine-binding protein (TBPA), transthyretin
- Sensitive marker of nutritional status (measured by neph/turb)
10
Q
Alpha 1 Proteins
A
- A1-Antitrypsin (AAT): protease inhibitor that binds/inactivates trypsin, deficiency associated w/ pulmonary emphysema & neonatal hepatitis, SPEP screening needs IEF to determine phenotype (ZZ & SZ high risk)
- A1-Acid Glycoprotein (AAG): APR, rarely measured
- A1-Fetoprotein (AFP): main fetal plasma protein, measured in amniotic fluid/maternal serum to screen for fetal abnormalities (neural tube defect), hepatic tumor marker
11
Q
Alpha 2 Proteins
A
- A2-Macroglobulin (A2MG): Largest non-Ig, increased in nephrotic syndrome- replaces lost albumin to maintain COP (not cleared, increased synthesis in liver), rarely measured
- Ceruloplasmin: Cu binding protein (8 Cu per molecule), plasma redox reactions (ferritin -> transferrin), screen for Wilson’s Dz
- Haptoglobin: binds Hgb irreversibly in intravascular hemolysis to prevent Fe loss (or bacteria use), low levels indicate hemolytic dz
12
Q
Beta Proteins
A
- Transferrin: Iron transport protein, increased in IDA (also pregnancy, estrogen therapy), decreased in inflammation/malignancy/liver dz, NEGATIVE APR
- C3/C4 Complement Proteins
13
Q
Complement Proteins
A
- Classical Pathway (C1-C4): Immune complexes recognized by C1, C2/C4 activate C3 (MAC)
- Alternative Pathway (C3, factor B/D, properdin): activated by bacteria, yeast, or endotoxins –> foreign surfaces
- C3 in both pathways, C4 classic only
14
Q
Complement Deficiency
A
- C2/C4**: Autoimmune Dz
- C3**: infection, encapsulated bacteria; low in all complement situations
- C5-C9: persistent Neisseria infection
- C1 Inhibitor: Hereditary Angioedema (HAE)–> can cause subcutaneous edema in laryngeal/bronchial/GI tissues, can be life-threatening, low C4
- CH50 = Total complement assay not preferred as large individual deficiencies may not be detected
15
Q
Immunoglobulins
A
- IgG: 2 heavy chains + 2 light chains, produced by plasma cells, long term immunity (or chronic dz), crosses placenta
- IgM: pentamer linked by J peptides, produced by plasma cells as first-line immunity and persists for 6-12months (acute dz)
- IgA: monomer or dimer, circulatory and secretory (tears, sweat, saliva etc), cleared by liver
NOT ON SPEP: - IgE: allergies
16
Q
Hypergammaglobulinemia
A
- Polyclonal: increase in multiple ig’s, seen as B-G smear on SPEP, IgG most obvious
- Monoclonal: single clone of plasma cells proliferates and produces single Ig (paraprotein or M-protein)
17
Q
Monoclonal Gammopathies
A
- Multiple Myeloma (60% of MGs)
- B-cell Lymphocytes (15%): Lymphoma, CLL
- Waldenstrom’s macroglobulinemia (IgM): hyperviscosity, Bence-Jones proteins
- MG of Undetermined Significance (MGUS): small MG w/o clinical symptoms, can progress in time and should be monitored
- Non-secretory MM: Multiple Myeloma that doesn’t produce Ig (detect by diff method)
- Serum Free Light Chains: deviate from 2:1 (K:L) ratio can indicate MG
18
Q
Multiple Myeloma
A
- Plasma cell malignancy, one or more clones (mainly IgG)
Commonly diffuse marrow distribution: - Plasmacytoma: solitary tumor
- Pancytopenia
- Osteolytic bone lesions: pain, fractures, breakdown by plasma cells
- Bence-Jones proteins found in urine (precipitates at 40-60C, re-dissolves at 100C)
19
Q
Cryoglobulins
A
Proteins that reversibly precipitate in temps below body temperature
Incubate at 4’ –> if precipitate forms and redissolves at 37’ –> Cryoglobulins are present
20
Q
Acute Phase Reactants (APR) + SPEP Pattern
Immediate response
A
Proteins whose concentration increase or decrease by >50% in response to tissue injury/inflammation/malignancy
- Positive APR: C3, C4, CRP, AAT, A2MG, Hpt, Fibrinogen
- Negative APR: Albumin, Pre-albumin, Trf
SPEP PATTERN:
- Albumin decrease (- APR)
- A2 increase due to Haptoglobin (+ APR)
21
Q
Delayed Response SPEP
A
- Albumin decreased (- APR)
- Haptoglobin increase (+ APR)
- Gamma globulin increase (Ab response)
22
Q
Nephrotic Syndrome SPEP
A
- Albumin decreases (renal loss)
- A2 increase (A2MG replacement of albumin)
- Gamma globulin decrease
23
Q
Hepatic Cirrhosis SPEP
A
- Albumin decreases (impaired synthesis)
- Beta-Gamma Bridging, Increased polyclonal gamma globulin (IgA increase, low hepatic clearing)
24
Q
Split-Beta SPEP
A
Separat ion of beta peak into two bands
- B1 = Transferring
- B2 = Complement C3
25
Monoclonal Gammopathy SPEP
- Mild albumin decrease
- Sharp increase in gamma region --> IgA MG may be seen as a sharp beak in A2/B region rather than gamma
- Beware of fibrinogen band in Beta region, can be mistaken for MG
26
Immunoelectrophoresis (IEF)
- Not commonly used anymore
- Sample added to cells in middle of gel, in between troughs
- Antisera to antigens of interest added to troughs
- If antigen is present in sample, precipitant arc will form in zone of equivalence after electrophoresis
- Thick and bulged arc indicates a positive reaction --> subjective and difficult to interpret
27
Immunofixation (IFE)
- Gel prepared to identify monoclonal gammopathies and light chain presence (G, A, M, Kappa, Lambda)
- Sample applied to slots and allowed to electrophorese, G/A/M/K/L antisera added to corresponding rows and allowed to precipitate
- Gel is stained and dried
- Positive gammopathy is where band is present in G/A/M and corresponding band in K/L (Ex. IgG - Kappa gammopathy)
- Possible to have biclonal or polyclonal gammopathies
28
Tumor Markers
- A substance produced by a tumor found in blood, body fluids, or tissue that may be used to predict the presence and size of the tumor, and/or monitor response to therapy
- Ideally, but not realistically useful in screening/diagnosis
- Main applications: predicting therapeutic response, detecting recurrence, monitoring treatment
29
Prostate-Specific Antigen (PSA)
- Enzyme tumor marker fairly specific to prostate tissue (hormonally regulated tissue, may be seen in breast)
- Exists free & bound to ACT & A2MG proteins
- Measurement enhancements --> age related reference intervals, PSA density (corrects for prostate volume), PSA velocity (change over time), Free PSA
* * PSA not considered to save lives, but enhanced measurements may be more clinically significant
30
Human Chorionic Gonadotropin (hCG)
- Hormone secreted by placenta during pregnancy (alpha proteins in early preg, beta in late); also seen in multiple tumor types
- Trophoblastic tumors: choriocarcinoma, hCG correlates well w/tumor volume, after surgery/chemo need hCG follow up yearly
- Phantom hCG: persistent low levels of hCG that can be due to residual tumor/HAMA,HAAA/heterophile Ab, doesn't show in urine hCG
- Assays: "Intact" measure alpha:beta dimer only, "Total" measure alpha:beta plus free beta (preferred for tumors, secrete more beta)
31
Oncofetal Antigens
Proteins produced during fetal life that decline or disappear at birth. They can reappear with cancer due to re-activation of transcription.
ex: AFP, CEA
32
Alpha-Fetoprotein (AFP)
- Major fetal plasma protein, increased in maternal serum during 3rd trimester
- Increased in liver disease, hepatocellular carcinoma (HCC, hepatoma) --> level correlates w/ tumor burden, useful in monitoring treatment
- With hCG, useful in classifying germ cell tumors (ie. Lance Armstrong had embryonal carc.)
33
Carcinoembryonic Antigen (CEA)
- Heterogenous glycoprotein in family of 36 proteins (10 genes)
- Elevated in many cancer types (Colorectal, GI, Panc, Lung, Breast/Ovarian/Uterine)
- Also elevated in non-cancerous conditions such as liver dz, IBD, pancreatitis
- Unlike hCG & AFP, levels correlate poorly with tumor burden
34
CA 15-3, CA 27.29
- Breast cancer markers (23%) but elevated in many other cancers, not good for screening
- Better for following dz/treatment: 25% increase is significant, increase may be due to tumor lysis in effective treatment
Assays:
- CA 15-3: dual monoclonal sandwich assay
- CA 27.29: single monoclonal assay
- Results are comparable but more information on CA 15-3
35
CA-125
- Marker for ovarian cancer: 50% S1, 90% S2, >90% S3/S4
- Also elevated in other cancers: endometrial, pancreatic, lung, breast, colorectal
- Not useful for screening due to non-specificity
- Can predict recurrent 2-3 mo before clinical symptoms
36
Other tumor markers
- Blood group antigens CA 19-9 & CA 72-4: Sialylated Lewis antigen & marker for pancreatic/colorectal cancer (19-9), not often used
- Immunoglobulins: markers of Multiple Myeloma
- Thyroglobulin: differentiated thyroid cancer, used to follow regeneration of thyroid tissue (not wanted following radiation)
- Chromogranin A: pheochromocytoma (adrenal medullary tumor)
