Plasma Proteins - Globulins and Protein Electrophoresis (not finished)

Question 1

What % of plasma proteins consists of fibrinogen

Answer 1

4%

Question 2

What does fibrinogen do?
(2)

Answer 2

It’s an important clotting factor

It’s converted into fibrin during the clotting process

Question 3

What are globulins

Answer 3

All non albumin proteins in blood except fibrinogen

Question 4

How do you calculate total globulins

Answer 4

Total globulins = Total protein - albumin

Question 5

Why do we measure total globulins

Answer 5

The total globulin fraction along with the albumin can be used to differentiate the causes of hypo and hyper proteinaemia

Question 6

Comment on the reference ranges for globulins
(3)

Answer 6

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

Question 7

What make up the globulin group

Answer 7

Gamma globulins

Enzymes

Carrier/transport proteins

Question 8

How does one determine the specific profile (constituents) of one’s globulins

Answer 8

This is done by serum electrophoresis

Question 9

What does serum electrophoresis

Answer 9

It separates blood proteins according to size and charge

Question 10

List the bands in an SPE (from left to right)

Answer 10

Albumin

Alpha-globulins

Beta-globulins

Gamma globulins

Question 11

Where are alpha and beta globulins made?
(2)

Answer 11

Made predominantly by the liver

They are increased during acute phase protein synthesis which occurs 2-5 days after injury to cells

Question 12

Where are gamma globulins made?

Answer 12

They are produced by plasma cells

Question 13

What causes an increase in total globulin and hyperproteinaemia

Answer 13

Dehydration

Immune response

Myeloma

Question 14

What causes a decrease in globulins
(6)

Answer 14

Liver disease (normal to low)

Renal disease

Salt retention syndromes

Intestinal malabsorption

Burns

Immune deficiency

Question 15

Define protein electrophoresis

Answer 15

A test that roughly quantitates the various protein fractions in serum

Question 16

In general what is the principle behind protein electrophoresis
(5)

Answer 16

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.

Question 17

How is electrophoresis done in the lab

Answer 17

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

Question 18

What are the five fractions of serum protein

Answer 18

Albumin
a1-globulin
a2- globulin
B-globulin
y-globulin

Question 19

Define electrophoresis
(4)

Answer 19

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

Question 20

Write about serum protein electrophoresis use

Answer 20

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

Question 21

Describe the albumin fraction

Answer 21

Narrow peak closest to the anode, has a strong negative charge

Question 22

Describe the a1-globulins

Answer 22

a1-acid glycoprotein
a1- antitrypsin
High density lipoprotein

Question 23

Describe the a2 globulins fraction

Answer 23

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

Question 24

Write about the B globulin

Answer 24

fibrinogen
C reactive protein – activates complement
protein C
complement
amyloid A
ferritin
LDL
IgM and IgA may bridge the β and γ regions

Question 25

What is found in the y1 globulin fraction

Answer 25

IgM
IgA
IgE

Question 26

What is found in the y2 globulin fraction

Answer 26

IgG

Question 27

Write about the clinical use of serum electrophoresis

Answer 27

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

Question 28

Write about gamma fraction SPE abnormalities

Answer 28

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

Question 29

What are monoclonal gammopathies

Answer 29

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).

Question 30

What indicates a monoclonal gammopathy

Answer 30

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).

Question 31

What might monoclonal gammopathies be associated with

Answer 31

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).

Question 32

Write about amyloidosis and monoclonal gammopathies

Answer 32

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.

Question 33

What are paraproteins
(4)

Answer 33

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

Question 34

What three conditions cause paraproteins
(3)

Answer 34

Myelomatosis accounts for most cases

Macroglobulinaemia = Waldenstrom’s Macroglobulinaemia

B-cell Lymphomas includes chronic lymphatic leukaemia

Question 35

What are referred to as “clonal”

Answer 35

Neoplastic proliferations of immunoglobulin-producing cells (plasma
cells and some B lymphocytes) are clonal.

Question 36

What are monoclonal peaks

Answer 36

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.

Question 37

What does the consequences of malignant B-cell proliferation depend on

Answer 37

Concentration of paraprotein
Presence or absence of immune paresis
Presence of BJP

Question 38

What four things might very high concentrations of paraproteins cause

Answer 38

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)

Question 39

What can presence of immune paresis mean

Answer 39

Abnormal spectrum of immunoglobulin may result in increased susceptibility to infection

Question 40

What can presence of Bence-Jones proteins mean

Answer 40

Renal glomerular dysfunction ->BJP deposited in tubular cells

Amyloidosis

Question 41

What is cryoglobulinaemia

Answer 41

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

Question 42

What are the two types of protein loss

Answer 42

Selective and non selective loss

Question 43

What is selective protein loss
(4)

Answer 43

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

Question 44

Write about non-selective protein loss

Answer 44

Due to either whole blood or serum loss, and all serum proteins are lost equally

Blood loss, burns, severe glomeular disease

Question 45

How do the glomeruli stop protein loss

Answer 45

Pore size
Negative charge on the basement membrane

Question 46

What causes protein in urine

Answer 46

Alteration of either pore size of negative charge on basement membrane of glomeruli

This may allow albumin and larger proteins to enter the filtrate

Question 47

What amount of protein in urine indicates disease

Answer 47

Proteinuria>0.15g/day

Question 48

What are the three types of proteinuria

Answer 48

Functional proteinuria
Postural proteinuria
Nephrotic proteinuria

Question 49

What is functional proteinuria

Answer 49

Seen with fever or exercise
<0.5g loss daily

Question 50

What is postural proteinuria

Answer 50

Seen with erect posture

Usually less than 1 gram/day

Question 51

What is nephrotic proteinuria

Answer 51

> 3.5g protein loss a day per 1.73 m2 surface area

Question 52

What are the three types of proteinuria

Answer 52

Overflow proteinuria
Tubular proteinuria
Glomerular proteinuria

Question 53

What is overflow proteinuria

Answer 53

Capacity to reabsorb protein from proximal tubule overwhelmed e.g. haemoglobinuria, myoglobinuria, Bence Jones proteinuria

Question 54

What is tubular proteinuria

Answer 54

Decreased tubular reabsorption of protein from
proximal tubule, always < 2 g daily; e.g. Fanconi
syndrome, tubulo-interstitial diseases, acute renal insufficiency, chronic hypokalemia.

Question 55

What is glomerular proteinuria

Answer 55

Causes of glomerular proteinuria include nil disease (minimal change disease) and other
glomerulonepathies.

Question 56

Write about the methods of urinary protein measurement
(4)

Answer 56

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

Question 57

Write about microalbuminuria

Answer 57

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.

Question 58

Why is microalbuminuria measured
(4)

Answer 58

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.

Question 59

What are the five key points about microalbuminuria

Answer 59

• 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

Question 60

What causes renal proteinuria

Answer 60

Increased glomerular permeability e.g. Nephrotic Syndrome

Albumin is usually the predominant protein in urine

Question 61

What is postural proteinuria

Answer 61

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

Question 62

What are the four characteristics of nephrotic syndrome

Answer 62

Proteinuria >5g/day
Hypoalbuminaemia
Oedema
Hyperlipidaemia