Laboratory Diagnosis of

Protein Abnormalities
Prepared by:
4th year medical students

Revised by:
Dr. Ramez Zaid & Dr. Rihab Ibrahim
Assistant Professors
Pathology Department
Faculty of Medicine

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 Introduction

■ Proteins are present in all body fluids. Plasma

proteins are the most frequently measured for
diagnostic purposes.

■ Serum proteins are synthesized in liver (mainly)

and plasma cells (mainly for Igs).

■ Over 100 individual proteins have a physiological

function in the plasma. 2
 Functions of serum proteins
1. Transport of nutrients, minerals, some drugs …
2. Osmotic regulation (by colloid oncotic pressure to
maintain normal blood volume - esp. by albumin).
3. Catalytic functions (enzymes).
4. Protective functions (Igs, complement,
antiprotease).
5. Blood clotting (clotting factors, thrombin, fibrinogen
… ).
6. Anticoagulant activity (plasminogen, antithrombin)
7. Buffering capacity (acid-base balance).
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4
Measurement of plasma proteins
■ Total serum protein levels (TSP) ~
6.5-8.5 g/dL

à Hyperproteinemia: TSP > 8.5 g/dL.

à Hypoproteinemia: TSP < 6.5 g/dL.

■ Quantitatively, the single most important

protein is albumin. Other proteins are known
collectively as globulins (except for
fibrinogen).
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■ TSP and Albumin are measured by direct
chemical methods in the lab. Then, all
fractions of globulins (mainly containing Igs)
are estimated by indirect ways:

▪ Globulins = TSP - Albumin.

▪ Albumin: Globulin ratio
(A:G ratio) = 1.5:1 - 3:1

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 Types of plasma proteins:
A.Albumin (3.5 - 5.0 g/dL): about 50% of TSP.
B.Globulins (2 - 3.5 g/dl).
- α − globulins: α1& α2−globulins.
- β − globulins: β1 & β2 globulins.
- γ _ globulins (about 50% of globulins).
c.Others (as fibrinogen … etc)

■Individual proteins in the α and β bands are relatively low, so

changes in their concentrations rarely affect TSP or total serum
globulins value à BUT they can be measured separately by
special techniques in certain situations.

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8
Serum Proteins Electrophoresis
■ Formerly widely used technique for the semi-quantitative
assessment of plasma proteins.

■ Protein in aqueous solution (as cellulose acetate or agarose

gel) are charged groups (because of carboxylic & amino
groups)
à So they can be separated under an electric field according
to their charge, size and media used.
à This well form distinct bands: albumin, α 1-and α 2-
globulins, β- globulins and γ- globulins.

■ Values and diagrams for each band can be estimated by

using computer programs.
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Normal serum proteins
electrophoresis

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 Immunofixation Electrophoresis

(IFE)
▪ A specific type of electrophoresis used to detect important
proteins using specific antigens.
à Most commonly used to differentiate Monoclonal
hypergammaglobulinemia (that show narrow gamma band on
serum electrophoresis) from Polyclonal
hypergammaglobulinemia (that show wide gamma band on serum
electrophoresis).
▪ So we use specific antigens for the heavy chains (Ig G, A, M, D &
E) and for the light chains (kappa and lambda) to prove the
mono-clonality of hypergammaglobulinemia, which is called M –
protein or paraproteinemia.
▪ This test is usually done on serum and urine.
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 Uses of protein electrophoresis
❑On serum:
-As a follow up to abnormal findings (such as TSP + albumin, elevated urine
protein levels, elevated calcium levels, or low white or red blood cell counts).
-When symptoms suggest an inflammatory condition, (autoimmune disease,
acute or chronic infection, a kidney or liver disorder, or a protein-losing condition).
-When symptoms suggest multiple myeloma (as bone pain, anemia, fatigue,
unexplained fractures, or recurrent infections). If sharp bands are seen à
Immunofixation electrophoresis is used to confirm monoclonality.
-Follow up of multiple myeloma patients.
❑On urine:

-To determine the cause of elevated urinary protein (as in nephrotic syndrome or
multiple myeloma …).
-When multiple myeloma is suspected.
❑On CSF:

-In multiple sclerosis to detect oligoclonal bands (multiple distinct bands in the CSF
seen in multiple sclerosis).
-Un-explained headaches or other neurologic symptoms to look for proteins suggestive
of inflammation or infection.

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 Changes in TSP

■Generally, it can be due to changes in any of three

factors: the rate of protein synthesis, the rate
of removal and the volume of distribution.

■Only changes in albumin (mainly) and

immunoglobulins will have a significant effect on
the total protein concentration.

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 Hypoproteinemia
➢ Mainly occurs due to changes in albumin.

➢ Mechanism:

- ↓ oncotic pressure à ↑ ECF à edema.

- ↓ Renal flow à ↑ renin, angiotensin &
aldosterone à ↑ Na retention à ↑
hydrostatic pressure à edema.

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 Causes of Hypoproteinemia:

■Notes:

(as Kwashiorkor disease) -Increased

-In
volume of distribution leads to relative decrease (Not a real decrease)
nephrotic syndrome TSP will be ↓ except for α2-macroglobulin which is not
lost in urine due to its large size.
-In liver failure TSP will be ↓ except for Ig A because it is synthesized in GI tract &

(esp. chronic , why?)

humoral immunodeficiencies

pregnancy , ascites

(as Ménétrier's disease)

Artifactual
Sample taken from drip line 15
 Hyperproteinemia:
àRarely seen in clinical practice.

Increased synthesis
hypergammaglobulinemia and paraproteinemia
Notes:
-As you can see TSP is affected by:
Decreased volume of distribution
dehydration
àPosture.
àSample procedure.
Artifactual àState of hydration.
over infusion of albumin àConcomitant diseases.
hemoconcentration as in:
using tourniquet
upright posture after a period of recumbency

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1. Albumin:
■The most abundant plasma protein.

■Albumin is a high-capacity, low-affinity transport protein for many

substances and drugs.
■It contributes for ~ 80% of the plasma oncotic pressure.

■Causes of ↑ and ↓ are discussed previously.

■Used as a test for assessing nutritional status and testing liver

function.
■Because of its long t ½ it is not useful in follow up for nutritional
recovery or in acute hepatitis à were albumin concentration is
almost normal (decreased in chronic).
■Molecular variants:

-Bisalbuminemia: A pair of albumin bands is seen on

electrophoresis, clinically insignificant.
-Analbuminemia: A rare, inherited condition (Albumin is less than
1 g/L). May cause episodic mild edema, but patients17are otherwise
well.
2. α1-Antitrypsin:
■Anti-protease.

■Its deficiency is associated with emphysema and liver

cirrhosis.
3. Haptoglobin:
■Binds free hemoglobin.

■↓ in intravascular hemolysis.

■↑ in acute phase response and in nephrotic syndrome.

4. α2-macroglobulin:
■Anti-protease.

■↑ in nephrotic syndrome.

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5. Ceruloplasmin:
■A copper-carrying protein.

■↓ in Wilson's disease.

6. Transferrin:
■Iron-transporting protein, normally 30% saturated.

■In hemochromatosis, 60% saturated.

7. Immunoglobulins:

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 Hypogammaglobulinemia

A. Physiological:
- Early in life Igs are low (except for Ig G, Why?) and ↑ with age.

B. Pathological:
➢Congenital (primary):

- X-linked agammaglobulinaemia (Bruton's disease), Selective IgA

deficiency, hyper-IgM with decreased IgG ...
➢Acquired (Secondary):
- Marrow replacement and inhibition leading to decreased number of
plasma cells as in malignant lymphoma, leukemia (↓ normal Igs) and
multiple myeloma (the abnormal monoclonal Ig ↑ while other Igs ↓
leading to recurrent infections.
- Protein-losing conditions (gut, kidney).

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 Hypergammaglobulinemia

A. Polyclonal hypergammaglobulinemia:
-Acute and chronic infections (all Igs are increased).

-Chronic liver disease (increase of IgA mainly due to decreased

catabolism + chronic hepatitis).
-Autoimmune diseases (as RA, SLE …).

-Malignancies.

B. Monoclonal hypergammaglobulinemia:
M-proteinemia or paraprotein:
A monoclonal immunoglobulin (identical heavy and light chain)
produced by a single clone of cells of the B-lymphocyte
(mostly plasma cells).

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➢ Multiple myeloma (MM):
- Paraproteinemia > 3 gram/dL (usually Ig G >> A >> M >>> E,D) with
osteolytic bone lesions, BM plasmacytosis, anemia, and hypercalcemia
… etc.

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➢ Monoclonal gammopathy of undetermined significance (MGUS):
- Seen in elderly > 70 yrs.

- Benign paraproteinemia
< 3 gram/dL (as in MM
but lower levels)
with no bone destruction
or other symptoms.

➢ Primary macroglobulinemia (Waldenstrom’s disease) :

- Paraproteinemia (mainly Ig M) leading to blood hyperviscosity
with increased risk of thrombosis. Neither bone destruction,
hypercalcemia nor anemia are present.
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Examples of abnormal electrophoresis:

à TSP & Albumin band

à α2-globulin band (due to

increased α2- macroglobulin)

à TSP & Albumin band

à γ-globulin (due to
chronic inflammation &
increased Ig A)

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à α1-globulins

à γ-globulin is almost
absent
(agammaglobulinemia)

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 Polyclonal hypergammaglobulinemia
(broad γ-globulin band)

Monoclonal hypergammaglobulinemia
(narrow spike like gamma globulin band – IgG, lambda28restricted)
 Acute Phase Response (APR)
■ It includes physiological changes that occur
following infection, inflammation, trauma,
burns … as:
- Hemodynamic changes.
- ↑ of coagulation and fibrinolytic activity.
- Leukocytosis.
- Pyrexia.
- Acute phase proteins or reactants;
à↑ or ↓ in certain plasma proteins concentration
by at least 25 %.

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■ Positive acute phase reactants:
- Plasma proteins that ↑ in APR.
- Mainly due to the effect of IL-6 on the liver (↑ synthesis).

■ Negative acute phase reactants:

- Plasma proteins that ↓ in APR.
- Resulted from increased vascular permeability.

+ve APR -ve APR

• •
• C-reactive protein (CRP) • Albumin
• Procalcitonin • Pre-albumin (transthyretin) *
• Serum amyloid A • Transferrin.
• Fibrinogen
• Ceruloplasmin
• α1-antitrypsin
• α1-acid glycoprotein (orosomucoid)
• α1-antichymotrypsin
• Haptoglobin
• C43 & C4 - complement 30
• Hemopexin
■ In APR the most striking pattern on electrophoresis is:
Acute à Increase in the α1 and α2 regions and mild
decrease in albumin.
Chronic à The same as above with an increase in
γ region ( due to Igs overproduction).

■ The decrease in –ve APR concentrations is very small,

so no significant changes on TSP will be seenà Not
used usually in practice.
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■ Acute phase reactants are used in:
1. Detection of inflammation (e.g. Rheumatoid arthritis).
2. Assess the intensity or activity of inflammation (either
acute or chronic) from the levels of acute phase
reactants.
3. Follow up and assessment of response to treatment
(for inflammation & tumors).

■ APR is most commonly assessed by measuring CRP

and ESR à Both are sensitive for inflammation but
not specific for any cause.

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1. C-reactive protein (CRP):
▪ Have a general function in defense against bacteria and foreign
substances.
▪ Can increase 30-fold from a normal value of less than 5 mg/L
during the APR.
▪ It is more useful, sensitive and more specific than measurement of
the ESR (esp. in acute inflammation).
▪ CRP begins to rise at about 6 h after the initiation of an acute phase
response and reaches a peak after about 48 h before beginning to fall.
▪ Significant increase in CRP is seen in bacterial infections. While
viral and some autoimmune diseases (as SLE & scleroderma) do not
usually cause a raised CRP.

■Disadvantages:

- Varies with age, sex and race.

- CRP of 5 – 10 mg/dL can be seen also in non-inflammatory states as:
Obesity, smoking, DM, HTN, IHD …

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2. Erythrocyte Sedimentation Rate (ESR):
■ It is the rate (mm/hr) at which RBCs suspended in plasma settle
when placed in a vertical tube à Common and simple test, BUT
less specific.
■ Normal Male Female (higher)
range Age in yrs / 2 (Age in yrs + 10) / 2

■ It is an indirect measurement of +ve APR in blood (esp.

fibrinogen).

■ Disadvantages:
- Changes in ESR is relatively slower than CRP.

- Influenced by number of RBCs (ESR increases in anemia).

- Increased with age, obesity, nephrotic syndrome and renal

failure. 34
Examples on the uses of CRP & ESR:
■ Acute inflammation (esp. bacterial):
- Septic arthritis.
- Pneumonia … etc.
■ Chronic inflammation:
- Rheumatoid arthritis (CRP is more useful).
- SLE (ESR is more useful).
- Polymyalgia rheumatica & giant cell arteritis.
- Chronic osteomyelitis.
■ “High sensitivity CRP” is a newly used test used in
the diagnosis & follow up of myocardial infarction.
■ Malignancies. 35
THANK YOU

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