Formic Acid Sodium Citrate Method 1. Decalcify for 5–14 days in formic acid-sodium citrate solution. Change solution daily for best results.

 

Principle

Immunoenzymometric Assay

The essential reagents required for an immunoenzymometric

assay include high affinity and specificity antibodies (enzyme

conjugated and immobilized), with different and distinct

epitope recognition, in excess, and native antigen. In this

procedure, the immobilization takes place during the assay

at the surface of a microplate well through the interaction

of streptavidin-coated on the well and exogenously added

biotinylated monoclonal anti-insulin antibody.

Upon mixing monoclonal biotinylated antibody,

the enzyme-labeled antibody and a serum containing

The Endocrine System 789

the native antigen, reaction results between the native

antigen and the antibodies, without competition or steric

hindrance, to form a soluble sandwich complex. The

interaction is illustrated by the following equation:

EnzAb(p) + AgIns. + BtnAb(m)

Ka

K-a EnzAb(p)-AgTSH-BtnAb(m)

BtnAb(m) = Biotinylated Monoclonal Antibody (Excess

Quantity)

AgINS. = Native Antigen (Variable Quantity)

EnzAb(p) = Enzyme labeled Monoclonal Antibody (Excess

Quantity)

EnzAb(p)-AgINS.-BtnAb(m) = Antigen Antibodies Sandwich

Complex

Ka = Rate Constant of Association

K-a = Rate Constant of Dissociation

Simultaneously, the complex is deposited to the well

through the high affinity reaction of streptavidin and

biotinylated antibody. This interaction is illustrated below:

EnzAb(p)-AgINS.-BtnAb(m) + StreptavidinC.W. ⇒

⇒ immobilized complex

StreptavidinC.W. = Streptavidin immobilized on well

Immobilized complex = Sandwich complex bound to the

solid surface.

After equilibrium is attained, the antibody bound

fraction is separated from unbound antigen by decantation

or aspiration. The enzyme activity in the antibodybound

fraction is directly proportional to the native antigen

concentration. By utilizing several different serum

references of known antigen values, a dose response curve

can be generated from which the antigen concentration of

an unknown can be ascertained.

Expected Values

Insulin values are consistently higher in plasma than in

serum; thus, serum is preferred. Compared with fasting

values in non-obese non-diabetic individuals, insulin

levels are higher in obese non-diabetic subjects and lower

in trained athletes. Although proinsulin cross reacts with

most competitive insulin assays, there is virtually less

than 0.01% cross reaction found with proinsulin using

Monobind Insulin Microwell CIA.

Each laboratory is advised to establish its own ranges

for normal and abnormal populations. These ranges are

always dependent upon locale, population, laboratory,

technique and specificity of the method.

Based on the clinical data gathered by Monobind in

concordance with the published literature the following

ranges have been assigned. These ranges should be used

as guidelines only:

Children < 12 years < 10 µIU/mL

Adult (Normal) 0.7 – 9.0 µU/mL

Diabetic (Type II) 0.7 – 25 µIU/mL

C-Peptide

Expected Values

C-peptide values are consistently higher in plasma than

in serum; thus, serum is preferred. Compared with fasting

values in non-obese non-diabetic individuals, C-peptide

levels are higher in obese nondiabetic subjects and lower

in trained athletes.

Anti-insulin

Clinical Relevance

Type 1 Diabetes is mainly characterized by limited or fully

missing secretion of the hormone insulin. Morphological

studies demonstrated a destruction of the beta cells of

the so-called Langerhans’ Islet Cells in Type 1 diabetics.

Numerous researchers described the appearance of

antibodies directed against the islet cells and insulin as the

causal reason for the onset of the disease.

Anti-insulin antibodies are found in 37% of patients

with newly detected Type I Diabetes, in 4% of their

relatives of the first degree and in up to 1-5% of healthy

controls. A positive correlation between the appearance

of anti-insulin and anti-islet cell antibodies has been

reported.

Anti-insulin autoantibodies may be detected several

months and in some cases years before the onset of the

fully clinical manifestation of the diseases. Occasionally

also autoantibodies to pro-insulin may appear.

These “true” anti-insulin autoantibodies directed against

endogenous insulin have to be distinguished from those

autoantibodies which are developed in insulin-dependent

diabetics undergoing therapy with insulin preparations of

animal origin. In fact, the latter have to be referred to side

effects. These side effects may occur as local reactions of

the skin by development of insulin-specific autoantibodies.

These autoantibodies are causing the formation of an

insulin depot and they may simulate a resistance against

the hormonal treatment with animal insulin.

790 Concise Book of Medical Laboratory Technology: Methods and Interpretations Autoantibody specificity type I diabetics

(%)

healthy controls

(%)

Anti-islet cell antibodies 32 1

Antibodies against islet cell

surface antigens approx.

50 2

Anti-insulin antibodies up to 70 0

Anti-thyroid peroxidase ab’s

(Anti-TPO)

18 6

Anti-single-stranded

DNA ab’s (Anti-ssDNA) 85 9

Additionally, other immunological phenomena

have been reported for Type I diabetics. A lot of other

autoantibody specificities have been d etected in those

patients too, but these antibodies must not cause

additional autoimmune phenomenon.

Indications

¾ Anti-insulin antibodies in Type I diabetics

¾ Development of anti-insulin antibodies under insulin

therapy.

Normal Values

In a normal range study with serum samples from healthy

blood donors, the following ranges have been established

with the anti-insulin test:

Anti-insulin [U/mL]

Normal < 5

Borderline 5–10

Elevated > 10

Positive results should be verified concerning the

entire clinical status of the patient. Also, every decision for

therapy should be taken individually.

25

Histopathology

C H A P T E R

PREPARATION OF TISSUES

Fixation

This is the process of killing and hardening. The first

phase of fixation is the rapid killing; the second phase, the

hardening of tissue. After removal, the tissue should be

put in the fixative immediately. The choice of a fixing agent

should be determined by the purpose of which the tissue

is to be stained or preserved. If several special stains may

be required, small blocks of the tissue should be fixed in

each of the following: 10% neutral formalin, Zenker’s fluid,

Bouin’s fluid and absolute alcohol or Carnoy’s fluid.

Blocks should be cut thin enough so that the fixing fluid

will penetrate the tissue in a reasonably short time. To do

this blocks should not be more than 0.5 cm thick and should

be immersed in at least 20 times their volumes of fixative.

Ten percent formalin is the most widely used fixative

because it is compatible with most stains. Length of

fixation depends on the size of blocks.

I. 10% Formalin solution

 37–40% Formaldehyde 100 cc

 Tap water 900 cc

II. Buffered neutral formalin solution

 37–40% Formaldehyde 100 cc

 Distilled water 900 cc

 Sodium phosphate monobasic 4 g

 Sodium phosphate dibasic 6.5 g

 III. Zenker’s fluid

 Distilled water 1000 cc

 Mercuric chloride 50 g

 Potassium dichromate 25 g

 Sodium sulfate 10 g

 Add 5 cc of glacial acetic acid to 95 cc of Zenker’s fluid

before use.

IV. Lugol’s solution (Weigert’s Modification)

 Potassium iodide 2 g

 Iodine 1 g

 Distilled water 100 cc

V. Bouin’s fluid

 Picric acid, saturated aqueous

 solution 750 cc

 37–40% Formaldehyde 250 cc

 Glacial acetic acid 50 cc

VI. Carnoy’s fluid

 Absolute alcohol 60 cc

 Chloroform 30 cc

 Acetic acid glacial 10 cc

VII. Absolute alcohol and acetone are also used as fixatives

for bacteria, glycogen, and some of the enzymes.

Decalcification

Bone and calcified tissue should be cut into small pieces

with a saw before fixation. After they are thoroughly fixed,

they are placed in a gauze bag tied with a string, which has

been dipped in melted paraffin. The bag is suspended in

a large quantity of decalcifying solution, at least a quart

for blocks of the average size. Stirring or agitation of the

fluid hastens decalcification. Every trace of decalcifying

solution must be removed by washing the pieces in

running water for several hours before dehydration and

embedding.

Nitric Acid Method

1. Decalcify sections in large quantities of 5% aqueous

solution of nitric acid for 1 to 4 days. Change the

solution daily. Sections of bone may be tested by

bending, piercing with a sharp needle or X-ray.

792 Concise Book of Medical Laboratory Technology: Methods and Interpretations

2. Wash in running water for 24 hours.

3. Neutralize in 10% formalin to which an excess of

calcium or magnesium carbonate has been added.

4. Wash in running water for 24 to 48 hours.

5. Dehydrate, clear and embed in either paraffin or

celloidin.

The method is used only for small pieces of bone, which

must be processed rapidly. Exposure of unduly long length

to nitric acid impairs or destroys nuclear staining.

Formic Acid Sodium Citrate Method

1. Decalcify for 5–14 days in formic acid-sodium citrate

solution. Change solution daily for best results.

Solution A

Sodium citrate 50 g

Distilled water 250 cc

Solution B

Formic acid (90%) 125 cc

Distilled water 125 cc

Mix solutions A and B for use

2. Wash in running water for 4 to 8 hours.

3. Dehydrate, clear and embed.

This technique gives better staining quality than the

nitric acid method.