Plasma Proteins - Globulins and Protein Electrophoresis (not finished) Flashcards
What % of plasma proteins consists of fibrinogen
4%
What does fibrinogen do?
(2)
It’s an important clotting factor
It’s converted into fibrin during the clotting process
What are globulins
All non albumin proteins in blood except fibrinogen
How do you calculate total globulins
Total globulins = Total protein - albumin
Why do we measure total globulins
The total globulin fraction along with the albumin can be used to differentiate the causes of hypo and hyper proteinaemia
Comment on the reference ranges for globulins
(3)
Males have a slightly higher total globulin than females
Children and neonates have slightly lower total globulin than adults
Some problems with supine measurements as with albumin and total protein
What make up the globulin group
Gamma globulins
Enzymes
Carrier/transport proteins
How does one determine the specific profile (constituents) of one’s globulins
This is done by serum electrophoresis
What does serum electrophoresis
It separates blood proteins according to size and charge
List the bands in an SPE (from left to right)
Albumin
Alpha-globulins
Beta-globulins
Gamma globulins
Where are alpha and beta globulins made?
(2)
Made predominantly by the liver
They are increased during acute phase protein synthesis which occurs 2-5 days after injury to cells
Where are gamma globulins made?
They are produced by plasma cells
What causes an increase in total globulin and hyperproteinaemia
Dehydration
Immune response
Myeloma
What causes a decrease in globulins
(6)
Liver disease (normal to low)
Renal disease
Salt retention syndromes
Intestinal malabsorption
Burns
Immune deficiency
Define protein electrophoresis
A test that roughly quantitates the various protein fractions in serum
In general what is the principle behind protein electrophoresis
(5)
Blood serum is placed on specially treated support and exposed to an electric
current.
The various proteins migrate (move on the support) to form bands that indicate the relative proportion of each protein
fraction.
Individual proteins, with the exception of albumin, are not usually measured.
However, protein fractions or groups ARE measured.
The levels of protein fractions can be roughly measured by measuring the total serum protein and multiplying by the relative percentage of each component protein fraction.
How is electrophoresis done in the lab
The procedure consists of applying a drop of serum to a support medium, such as a sheet of cellulose acetate or agarose that is soaked in a buffer in a chamber.
In this alkaline environment, all the proteins have a negative charge but each has a different magnitude.
As an electric current is passed through the medium, the proteins migrate toward the positively charged anode and are separated because of their charge differences into several bands on the medium.
After a set period, the cellulose acetate sheet with the separated proteins is removed, fixed, cleared, and the proteins stained.
The staining reveals a series of bands at different positions; the width and density of the bands depends on the amount of proteins with that particular electrophoretic mobility
What are the five fractions of serum protein
Albumin
a1-globulin
a2- globulin
B-globulin
y-globulin
Define electrophoresis
(4)
A method of separating proteins based upon the charge and molecular weight of the protein.
Most proteins are negatively charged and will move toward a positive electrode (anode).
The amounts and locations of the separated proteins are represented graphically.
The concentration of each protein peak can be determined by calculating the area under the curve on the electrophoretogram
Write about serum protein electrophoresis use
SPE is an important screening test for serum protein abnormalities.
Samples are applied to a gel prior to electrophoresis and staining.
Sera showing abnormal results should be further investigated by Immuno-Fixation Electrophoresis [IFE].
Serum sample is preferred to Plasma sample
Serum sample contains all Plasma proteins minus clotting agents [Notably Fibrinogen]
Haemolysed samples can cause problems
Describe the albumin fraction
Narrow peak closest to the anode, has a strong negative charge
Describe the a1-globulins
a1-acid glycoprotein
a1- antitrypsin
High density lipoprotein
Describe the a2 globulins fraction
Haptoglobin - largest portion of this peak, binds free Hb
Serum amyloid A
a2- macroglobulin - proteinase inhibitor
Caeruloplasmin
Very low density lipoprotein (VLDL)
Low density lipoproteion
Write about the B globulin
fibrinogen
C reactive protein – activates complement
protein C
complement
amyloid A
ferritin
LDL
IgM and IgA may bridge the β and γ regions
What is found in the y1 globulin fraction
IgM
IgA
IgE
What is found in the y2 globulin fraction
IgG
Write about the clinical use of serum electrophoresis
The main diagnostic indication for performing serum protein electrophoresis (SPE) is to get more information about the protein or proteins that are increased in a patient with hyperglobulinemia, which is detected in the chemistry panel.
Routine clinical chemistry panel includes a measurement of total protein and albumin and the total globulin result is the difference
between these two measured values.
SPE is of most help in distinguishing hyperglobulinemia caused by
inflammation and/or immune response from hyperglobulinemia caused by the neoplastic proliferation of a clone of antibody
producing cells, i.e. multiple myeloma.
This procedure is usually not indicated unless the globulin result in the chemistry panel is
very high, e.g. > 50-60 g/L.
Hepatic synthesis [Liver] of many different proteins is stimulated by cytokines secreted by cells at sites of inflammation or tissue injury.
These proteins, called acute phase reactants (APR), are alpha globulins, so a larger than
normal peak in the alpha 1 and/or alpha 2 region is a sign of inflammatory disease.
Increase in alpha globulins is detectable very soon after onset of inflammation and persists until the inflammation is resolved.
Some APR proteins migrate in beta and gamma regions and can contribute to increases in these peaks
Write about gamma fraction SPE abnormalities
Immunoglobulins [Globulins] migrate in the gamma and beta regions (IgG is in the gamma peak; IgA and IgM extend into the
beta peak).
Immunoglobulins produced by lymphocytes and plasma cells in immune responses are of different specificities and immunoglobulin classes and migrate to slightly different points during electrophoresis, forming peaks in the beta and gamma regions that are taller than normal and wider than the albumin peak at both the base and the tip.
Such broad-based peaks in the gamma region are described as polyclonal
What are monoclonal gammopathies
Are myeloproliferative disorders which constitute a group of diseases characterized by the proliferation of a single clone of plasma cells or B-lymphocytes that produce a
homogeneous monoclonal protein (M-protein: M-component; monoclonal Ig-band or paraprotein).
What indicates a monoclonal gammopathy
The M-protein, which may be a polymer, monomer or fragment of an immunoglobulin or only free light chains, is recognized as a discrete band of restricted migration – a narrow discrete spike – on electrophoresis.
* When the band represents a monoclonal free light chain, it usually is called a Bence Jones protein (BJP).
What might monoclonal gammopathies be associated with
malignant proliferations of lymphocytes or plasma cells, i.e., the B-cell malignancies, including multiple myeloma (MM), and Waldenström’s macroglobulinemia (WM)
Benign disease such as the monoclonal gammopathy of undetermined significance (MGUS).
Write about amyloidosis and monoclonal gammopathies
Primary (AL) amyloidosis may be associated with multiple myeloma and rarely with lymphoid malignancies, but most cases can be considered as a particular form of monoclonal gammopathy, where the monoclonal free light chain causes damage by virtue of its amyloidogenic properties.
What are paraproteins
(4)
Refers to the presence of an abnormal, narrow, dense band on the electrophoretic strip.
Commonly found in the g -region but may be seen anywhere from the a 2 to g region.
A paraprotein can often be shown to be monoclonal.
The presence of a paraprotein is strongly suggestive, it is not diagnostic of a malignant process
What three conditions cause paraproteins
(3)
Myelomatosis accounts for most cases
Macroglobulinaemia = Waldenstrom’s Macroglobulinaemia
B-cell Lymphomas includes chronic lymphatic leukaemia
What are referred to as “clonal”
Neoplastic proliferations of immunoglobulin-producing cells (plasma
cells and some B lymphocytes) are clonal.
What are monoclonal peaks
The immunoglobulin produced by a single clone of B lymphocytes or plasma cells is very homogeneous and typically forms a peak in the gamma region that is as narrow at base and tip as the albumin
peak.
Such peaks are described as monoclonal.
What does the consequences of malignant B-cell proliferation depend on
Concentration of paraprotein
Presence or absence of immune paresis
Presence of BJP
What four things might very high concentrations of paraproteins cause
In vivo effects of increased plasma viscosity (sluggish flow) can cause retinal-vein thrombosis, peripheral gangrene
May be noticed during venepuncture (blood films difficult)
A high plasma total protein concentration, with normal or low albumin
Spurious hyponatraemia (psudohyponatraemia)
What can presence of immune paresis mean
Abnormal spectrum of immunoglobulin may result in increased susceptibility to infection
What can presence of Bence-Jones proteins mean
Renal glomerular dysfunction ->BJP deposited in tubular cells
Amyloidosis
What is cryoglobulinaemia
Proteins that precipitate when cooled below body temperature
May be associated with disease which produce paraproteins
Half of these proteins are monoclonal immunoglobulins and the rest are polyclonal immunoglobulin complexes
Usually present with other symptoms of underlying disease
Intravascular precipitation may occur above 22 degrees and patient may have skin lesions and Raynaud’s phenomenon
Benign paraproteinaemia present in up to 30% of cases
What are the two types of protein loss
Selective and non selective loss
What is selective protein loss
(4)
Loss through a semipermeable membrane or tight intracellular channels
Proteins are lost in the urine in concentrations inversely proportional to their molecular size.
Small proteins pass through
the kidney and into the urine in high concentrations, while large proteins pass through the kidneys minimally, if at all.
As a result, serum concentration of small proteins declines, while those of larger proteins remains the same or increases
Write about non-selective protein loss
Due to either whole blood or serum loss, and all serum proteins are lost equally
Blood loss, burns, severe glomeular disease
How do the glomeruli stop protein loss
Pore size
Negative charge on the basement membrane
What causes protein in urine
Alteration of either pore size of negative charge on basement membrane of glomeruli
This may allow albumin and larger proteins to enter the filtrate
What amount of protein in urine indicates disease
Proteinuria>0.15g/day
What are the three types of proteinuria
Functional proteinuria
Postural proteinuria
Nephrotic proteinuria
What is functional proteinuria
Seen with fever or exercise
<0.5g loss daily
What is postural proteinuria
Seen with erect posture
Usually less than 1 gram/day
What is nephrotic proteinuria
> 3.5g protein loss a day per 1.73 m2 surface area
What are the three types of proteinuria
Overflow proteinuria
Tubular proteinuria
Glomerular proteinuria
What is overflow proteinuria
Capacity to reabsorb protein from proximal tubule overwhelmed e.g. haemoglobinuria, myoglobinuria, Bence Jones proteinuria
What is tubular proteinuria
Decreased tubular reabsorption of protein from
proximal tubule, always < 2 g daily; e.g. Fanconi
syndrome, tubulo-interstitial diseases, acute renal insufficiency, chronic hypokalemia.
What is glomerular proteinuria
Causes of glomerular proteinuria include nil disease (minimal change disease) and other
glomerulonepathies.
Write about the methods of urinary protein measurement
(4)
Screening test: Dye-impregnated paper stips
24 hour protein excretion
Spot urine protein/creatinine ratio
24-hour urine collection for albumin or spot albumin to creatinine ratio if dip stick is negative
Write about microalbuminuria
The early signs of diabetic nephropathy cannot be detected by the routine screening tests for proteinuria so that more sensitive methods for detecting abnormal albumin excretion must be used.
- The early stage of albuminuria (microalbuminuria) is clinically defined as an albumin excretion rate of 30-300 mg/24 hours (20-200 μg/min).
- An elevated albumin excretion rate of >30-300 mg/24 hours (>20-200 μg/min; macroalbuminuria) is an indicator of cardiovascular risk in all subjects and especially in patients with metabolic syndrome and in people with type 2 diabetes.
Why is microalbuminuria measured
(4)
Identifies patients at risk for the development of diabetic nephropathy, hypertension and both chronic renal disease and CVD.
- The risk of progression to renal disease in patients with microalbuminuria is 20-fold higher than in patients with normal excretion.
- Elevated blood pressure in type 2 patients is the major predictive factor for the development of microalbuminuria.
The finding of microalbuminuria in 35-40% of diabetes type 1, 25-60% of type 2 and in 10-15% of those without diabetes is associated with an increased relative risk for future cardiovascular events, including myocardial infarction (MI), stroke, cardiovascular death and CHF hospitalization.
What are the five key points about microalbuminuria
- First clinical sign of diabetic nephropathy
- Incidence increases with duration of diabetes - may be present
at diagnosis in NIDDM - Transient albuminuria may occur with fever, infection, exercise,
heart failure - Persistent or increasing albuminuria increases the risk of
development of overt nephropathy by 20-fold. - Associated with poor glycemic control, elevated blood pressure
What causes renal proteinuria
Increased glomerular permeability e.g. Nephrotic Syndrome
Albumin is usually the predominant protein in urine
What is postural proteinuria
More severe proteinuria in the upright prone position, may disappear at night.
Commonest in adolescents and young adults, often harmless but may be a sign of renal disease in later years
What are the four characteristics of nephrotic syndrome
Proteinuria >5g/day
Hypoalbuminaemia
Oedema
Hyperlipidaemia
