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Serum and Plasma

Centrifugation
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PLASMA PROTEINS
PROF.S.O.OLUSI

Introduction

Proteins are present in all body fluids but it is the protein of the blood
plasma that are examined most frequently for diagnostic purposes.
There are more than 100 individual plasma proteins.The most
important plasma protein is albumin.The other plasma proteins
are known collectively as globulins.

Total protein = Albumin + Globulins


Where do plasma proteins come from?

The amount of protein in plasma depends on the balance between the


rate of synthesis and that of catabolism or loss.

Synthesis
- most proteins - hepatocytes
- complement - liver and macrophages
- immunoglobulins - β cells of the immune system

Catabolism and loss


- most plasma proteins are catabolised by capillary endothelial
cells or mononuclear phagocytes, or renal tubular cells
- loss occurs in the kidneys and GIT.
Principal Plasma Proteins

Class Protein Approx. mean serum


concentration (g/L)
prealbumin 0.25
albumin 40
α1 -globulins α1 – antitrypsin 2.9
α1 – acid glycoprotein 1.0
haptoglobins 2.0
α2- globulins α2 - macroglobulin 2.6
caeruloplasmin 0.35
transferrin 3.0
low density lipoproteins 1.0
β-globulins
complement components 1.0
IgG 14.0
IgA 3.5
IgM 1.5
γ globulins IgD 0.03
IgE trace
Functions of Plasma
Proteins
Function Example
Transport thyroxine-binding globulin (thyroid hormones)
apolipoproteins (cholesterol, triglyceride)
transferrin (iron)

humoral immunity immunoglobulins

maintenance of all proteins, particularly albumin


oncotic pressure

Enzymes renin
clotting factors
complement proteins

protease inhibitors α1 – antitrypsin (acts on proteases)

buffering all proteins


Methods of assessing plasma proteins

1.Quantitation(dye binding methods)- Total proteins


- Albumin
2. Calculation : Globulin = Total proteins - albumin
3. Nephelometric quantitation : individual plasma proteins
4. Immunoassays : individual plasma proteins especially IgD and IgE
5. Electrophoresis : Electrophoresis, which separates proteins
according to their different electrical charges, is usually performed
by applying a small amount of serum to a strip of cellulose acetate
or agarose and passing a current across it for a standard time.Five
major groups of proteins albumin, α1, α2 , β and γ globulins are
separated.Each band can be quantified by densitometry.
Use of Plasma Proteins in
Diagnosis

A. Plasma Total Protein concentration


The normal plasma total protein concentration is 65 - 80 g/L

Low levels
♦ dilution as in excess fluid (over hydration)
♦ hypoalbuminaemia
♦ profound immunoglobulin deficiency

High levels
♦ loss of protein-free fluid-dehydration
♦ excessive stasis during venepuncture
♦ increase in one or more of the immunoglobulins
B. Albumin

Albumin, the most abundant plasma protein, makes the major


contribution (about 80%) to the oncotic pressure of plasma

Causes of hypoalbuminaemia

Decreased synthesis
malnutrition
malabsorption
liver disease

Increased volume of distribution


over-hydration
increased capillary permeability :
septicaemia
hypoxaemia
Increased excretion/degradation
nephrotic syndrome
protein-losing enteropathies
burns
haemorrhage
catabolic states :
severe sepsis
fever
trauma
malignant disease

Consequences of hypoalbuminaemia
♦ oedema
♦ loss of binding functions eg. Calcium, bilirubin, drugs, FFA
α 1 - Antitrypsin

This α1-globulin is a naturally occuring inhibitor of proteases.Its significance is


related to the clinical consequences of inherited disorders of α1 - antitrypsin
synthesis.These can cause emphysema, occuring at a yonger age (third and
fourth decades)than is usual for this condition, and neonatal hepatitis which
can progress to cirrhosis.

Haptoglobin

Haptogolubin is an α2- globulin.Its function is to bind free haemoglobin


released into the plasma during intravascular haemolysis.The haemoglobin-
haptoglobin complexes formed are removed by the reticuloendothelial system
and the concentration can be indicative of intravascular haemolysis.However,
low concentrations due to decreased synthesis are seen in chronic liver disease,
metastatic
disease and severe sepsis
α 2 -Macroglobulin
α2 -Macroglobulin is a high molecular weight protein (820,000 daltons) that
constitutes approximately one - third of the α2 -globulins.Its increased
synthesis in hypoalbuminaeemic states contributes to the increased α2
-globulin band seen on electrophoresis.Like α1 - antitrypsin, α2 -macroglobin
is an inhibitor of proteases, though it has a broader spectrum of activity.

Caeruloplasmin
A deficiency of this copper-carrying α2 -globulin is characteristic of
Wilson’s disease.Its concentration is increased in pregnancy and by oestrogen-
containing oral contraceptives.It is also an acute phase protein.

Transferrin
This β-globulin is the major iron-transporting protein in the plasma;normally
about 30%saturated with iron, it is characteristically saturated with iron in
haemochromatosis.Its measurement maybe useful in the assessment of a
patient’s response to nutritional support
Plasma Immunoglobulins
Properties and Functions of Plasma Immunoglobulins
Immunoglobulins are proteins which have antibody activity.There are
five classes (G, A, M, D, E), based on the type of heavy chain present.
Hypergammaglobulinaemia

Polyclonal increase (diffuse)


♦ chronic infections
♦ chronic liver disease (eg. cirrhosis of the liver)
♦ autoimmune disease (eg. rheumatoid arthritis)
♦ sarcoidosis

Monoclonal increase (paraprotein, M band)


Malignant ♦ mylomatosis
♦ Waldenstrom’s macroglobulinaemia
♦ heavy chain disease
♦ lymphoreticular malignancy
Hypogammaglobulinaemia

A. Decreased synthesis
♦ transient (neonate/infant)
♦ primary defective immune system
♦ secondary - malignancy
- uraemia
- diabetes mellitus
- Cushing’s syndrome
- AIDS

B. Protein Loss ♦ nephrotic syndrome


♦ protein- losing enteropathy
PARAPROTEINAEMIAS

Definition : Paraproteins are abnormal immunoglobulins or


their fragments found in blood, urine and tissues and
produced by abnormal B-lymphocytes.

Classification : Paraproteins are found in serum and / or


urine of many individuals and are associated with both
malignant and non-malignant conditions.
Malignant Paraproteinaemias

♦ Multiple myeloma

♦ Plasmacytoma
♦ Waldenstrom’s macroglobulinaemia
♦ Lymphoma
♦ Chronic Lymphatic Leukaemia
Symptomatic Non-Malignant Paraproteinaemias

♦ Amyloidosis
♦ Cryoglobulinaemiaa / immune complex disease
♦ Primary cold agglutinin disease

Asymptomatic Non-Malignant Paraproteinaemias

♦ Benign
♦ Transient
Other Diseases

♦ Peripheral Neuropathy

♦ Lichen myxoedermatosis

♦ Pyoderma gangrenosum

♦ Erythema elevatum diutinum


Findings that may lead to investigation for Paraproteinaemia

Clinical Symptoms
♦ Back pain
♦ Fatigue
♦ Recurrent infections

Clinical Syndromes

♦ Nephrotic
♦ Peripheral neuropathy
♦ Hyperviscosity
♦ Carpal tunnel
♦ Malabsorption
Radiological Findings
♦ Osteolytic lesions
♦ Pathological fractures

Haematological Findings

♦ Normochromic, normocytic anaemia


♦ Raised ESR
Biochemical Findings

♦ Raised serum total protein


♦ Hypercalcaemia
♦ Abnormal renal function tests
♦ Proteinuria
♦ Abnormal serum immunoglobulin concentrations
Laboratory investigation of Paraproteins

♦ Identification

Serum Protein Electrophoresis

♦Typing of Paraprotein

Immunofixation
MULTIPLE MYELOMA

Multiple myeloma is the commonest malignant cause of paraproteinaemia


It is a disease of the elderly (mean age 55 years ), with a peak incidence
between 70 and 75 years.Common presenting features are bone pain,
hypercalcaemia, fever, renal failure and infection.

Multiple myeloma is defined by three main features :


♦ the prescence of paraprotein
♦ the prescence of abnormal plasma cells in the bone marrow
♦ the destruction of bone
A diagnosis of MM can be made when any two of the features are present

Investigations :
♦ Identification and typing of paraprotein in serum and urine
♦ Bone marrow biopsy
♦ Radiological examination of bone
Laboratory investigations in myeloma
PROTEINURIA
Measurement of proteinuria plays an essential role in the assessment of renal
function.The presence of proteinuria is the hall mark of most renal diseases.
Protein appears in the urine from six different causes.

1. Change in glomerular permeability to plasma proteins – the most common cause


proteinuria.

2. Change in tubular reabsorption of normally filtered plasma proteins – results


in a modest excretion of proteins with molecular weights from 10,000 to 70,000
daltons.

3. Prerenal formation and glomerular filtration of paraprotein or endogenous


proteins [i.e.,Bence Jones proteins in multiple myeloma,myoglobin in crush
injuries or hemoglobin in hemolytic transfusion reactions, fibrin split products
in disaeminated intravascular coagulopathy(DIC) – referred to as
overproduction protenuria.

4. Increased tubular secretions, usually Tamm- Horsfall protein, urokinase ,sIgA

5. Release of renal tissue and tissue products e.g LDH,GGT

6. Obstruction of renal lymphatics, which leads to chyluria.


CLASSIFICATION OF PROTEINURIA
On the basis of electrophoretic and immunochemical analysis, proteinuria can be
classified into 3 types:

 Glomerular Proteinuria – presence of large quantities of high


molecular weight proteins especially albumin. This is indicative of
glomerular injury. Glomerular proteinuria may be mild, moderate, or
heavy depending on the natures of the disorder. Nephrotic
syndrome is diagnosed when protein excretion exceeds 3.5g/day.

Tubular Proteinuria – excretion of low – molecular weight proteins that,


are normally reabsorbed by renal tubules such as β 2M, alpha2 microglobulin
ribonuclease, lysosome and insulin,NAG,Urokinase
Protein excretion in tubular disorders rarely exceeds 2g/day.

Causes: renal tubular acidosis, Fanconi syndrome, pyelonephritis,


medulary cystic disease.

 Overproduction Proteinuria – protenuria is nonrenal disorders due


to the overproduction and excretion of a specific protein eg.
BenceJones proteins in multiple myleoma
Other types of Proteinuria:

i. Intermittent or transient proteinuria occurs after heavy exercise, fever or


emotional stress. This is considered to be a functional proteinuria and is normal.

ii. Postural or Orthostatic Proteinuria: Increased excretion of protein while in the


upright position and normal excretion in the recumbent position. Occurs in the
healing phase of many glomerular disorders. The total daily excretion of protein
rarely exceeds 1g in these patients.

Microalbuminuria : Normal daily excretion of albumin is less than 50mg. Patients


who excrete between 50 - 150mg/day but whose total urine protein excretion is
normal are said to have microalbuminuria. It has been found to be a reliable
indicator of the subsequent development of overt diabetic nephropathy or mortality
from renal or cardiovascular disease. Microalbuminuria is measured either by
Nephelometry or ELISA.
Laboratory Assessment of Proteinuria

a. Qualitative or semiquantitative urine Protein Testing :

• On random urine specimens is part of urinalysis.


• Dipstick testing is based on the principle of protein error of indicators. It lacks
sensitivity because below 30mg/dL, the dipstick test is unreliable. Dipstick
sensitivity to proteins other than albumin is also unreliable.

b. Determination of protein level in 12 – 24 hour urine collection

This is more accurate. Reference ranges for total urine protein in a normal
24 hour specimen is 50 – 300mg/day.

c. Urine albumin to creatinine ratio on a random sample of urine.

First morning specimens are not recommended because of the variation in


protein excretion in the recumbent and upright positions.
Stepwise investigation of patients with proteinuria
Protein in Serous Fluids
The lungs, heart and
abdominal cavity are
surrounded by single -
celled membranous, bi-
layered sacs permeable to
serum constituents. When
serum dialyses across these
membranes, the fluid
formed is called serous fluid;
Transudate / Exudate

The classification of serous fluid as


transudate or exudate is important.
Transudates are secondary to distant
pathology and indicate that treatment
should begin elsewhere. An exudate
indicates primary involvement, such as
infection and demands immediate
attention. The classification of a fluid as a
transudate or exudate is based on the
Light’s criteria (Fluid/Plasma ratios). If the
F/P for total protein is greater than 0.5,
and that for lactate dehydrogenase is
greater than 0.6, the fluid is an exudate. A
S/F albumin ratio equal to or greater than
1.1 usually indicates a transudate.