Operator Errors

Errors of the operator can be minimized by the practice

of good technique in the laboratory preparation of the

solutions and in the operation of the instrument. The

operator should be guided in the latter instance by the

instructions provided by the manufacturer. An awareness,

of the sources of error in the preceding categories should

tend to reduce these errors.

CLINICAL CHEMISTRY

Specimen Collection and Processing

With the exception of glucose, triglycerides and inorganic

phosphorus, most blood chemical constituents reveal no

significant change after a standard breakfast, so it is not

essential for the patient to be in an absolute fasting state

prior to blood specimen collection. However, lipemia

(lactescence), caused by transient rise in triglycerides

as chylomicrons following a meal containing fat may

cause interference with a large number of chemical

determinations because of turbidity. Therefore, blood

is always collected from a patient in the post-absorptive

state. This can be accomplished with an overnight fast

(12–14 hours, especially for lipids), although a 4 to 6 hours

fast will usually suffice.

Venipuncture should be performed for obtaining blood.

Disposable needles eliminate the hazard of serum hepatitis

transmission. Heparin is the most ideal anticoagulant for

plasma determination. The cost is quite prohibitive so

others EDTA, and trisodium citrate can be used without

significant alteration in reading and results. For glucose,

oxalate-fluoride mixture is used. Fluoride impairs

glycolysis of the blood cells. Prompt separation of plasma/

serum is essential to yield a proper specimen for most

chemical estimations. Always collect a little more blood

than required so as not to fall short of it subsequently. For

1 mL of serum about 2.5 mL of blood should be withdrawn.

Labelling and identification is important.

Pipettes: For dispensing test materials or reagents, etc. exact

quantities are needed. For volumes till 0.1 mL Borosil’s

pipettes can be used. Otherwise autopipettes for micro to

macro sampling can be used. Dispensing exact amounts

of test samples/reagents is the first step to accurate clinical

chemistry.

Proper Specimen Collection

If the commercially available kits are being used, follow

the manufacturers guidelines and collect the requisite

amount of blood.

Specimen Collection

Chemistry (plain tube)

Amylase Lipase

Alcohol Lithium

Bilirubin LATS and TSH

Barbiturate Triglyceride

Salicylate Electrolytes

BSP BUN

Calcium Uric acid

Cholesterol

Copper

Creatinine

CPK

SGOT

SGPT

Urea

T4, T3, TSH

Iron and iron binding

capacity

LDH

Chemistry (Heparin)

pH

Ammonia

RBC Potassium

Renin

Plasma testosterone

Cholinesterase

Plasma cortisol

Methemoglobin

Plasma hemoglobin

Chemistry

Oxalate, fluoride tube

Glucose

Glucose tolerance test

466 Concise Book of Medical Laboratory Technology: Methods and Interpretations Hematology (EDTA)

Complete blood counts

WBC, RBC, Hb, PCV,

MCV, MCH, MCHC

Differential count

Absolute eosinophil count

Hematology (EDTA)

Hb electrophoresis

G6PD screening

Reticulocyte count

ESR

Sickling test

Platelet count.

Hematology (plain tube)

Haptoglobin,

LE preparation

Serum viscosity.

Hematology (Sodium citrate)

PTTK

Prothrombin time

Thrombin time

Fibrinogen titer

Fibrinogen level.

Blood bank (plain tube)

Crossmatch

Typing

Coombs’ test

Antibody identification.

Serology (plain tube)

α1 antitrypsin

Antinuclear antibody

Antistreptolysin-O

Antithyroid antibodies

Ceruloplasmin

C-reactive protein

Cold agglutinins

Paul Bunnel test

Immunoglobulins

Leptospira agglutination test

VDRL

Australia antigen.

Processing

Ideally all measurements should be performed within

1 hour after collection. Tests where proteins are first

precipitated with tungstic acid, trichloroacetic acid or

barium sulfate—samples for these tests can be stored in

a refrigerator at 4–6°C if the interval before the analysis

exceeds 30 minutes. In medical chemistry, plasma can be

used for virtually all measurements (ideal anticoagulant

being heparin), although a few require serum (serum

enzymes and protein electrophoresis), while whole blood

can for all practical purposes be eliminated. Whenever a

delay of more than 1 hour is anticipated, refrigerate the

sample at 4oC. For extracting serum—let the blood clot at

room temperature (takes about 20–30 minutes), loosen the

clot at the top by a stick. Centrifuge blood for 10 minutes

at 3,000 rpm, serum can be removed with the use of a

pasteur pipette. Label and store the serum in a refrigerator

at 4–6°C until analyzed or freeze at –20°C, if the analysis is

to be delayed by more than 4 hours.

Centrifuge

While centrifuging the principle of balance must always be

observed. Tubes of equal weight, shape, and size should be

placed in opposing positions in the centrifuge head (using

water filled tubes whenever necessary). Tubes should be

supported by appropriately shaped rubber cushions in the

carrier of the centrifuge head. The speed of the centrifuge

should be slowly accelerated.

Difficulties

1. All tubes should be chemically clean, i.e. free of actual

or potential organic and/or inorganic constituents that

may alter the result of a chemical analysis. They need

not be sterile.

2. Hemolysis: It should always be avoided as release of RBC

contents (e.g. LDH, acid phosphatase and potassium)

or through color change (especially for photometric

measurements using shorter wavelengths of the visible

spectrum 400–500 nm) results may be falsely high.

Hemoglobin interferes with specific chemical reactions

(e.g. diazotization inhibition in bilirubin estimation).

Blood Collection, Precautions and Errors

1. Excessive venous stasis by prolonged application

of tourniquet should be avoided. This would also

raise concentration of certain constituents of blood

hormones, calcium, K+, Lactic acid, etc.).

2. The syringe, needle and the tube should be moisture

free.

3. Blood should be withdrawn by needle of gauge less

than 21.

4. Expelling blood through the needle into the container

should be avoided.

Clinical Chemistry 467

5. Do not shake blood in container to mix with anticoagulants. Mix by gentle repetitive inversion—about

6 to 8 times.

 

4. Elastase: Digests elastin, which is the most resistant of

all body proteins to lytic agents.

Peptidases

1. Carboxypeptidase: The active enzyme removes amino

acids one by one from the carboxyl ends of the peptide

chains.

2. Aminopeptidase: The active enzyme removes amino

acids one by one from the ends of the peptide chains

bearing the free amino groups.

Nucleases

Ribonuclease and deoxyribonuclease are secreted in

probably more than one or perhaps several forms, they

hydrolyze the respective nucleic acids.

Amylolytic Enzymes

Amylase: Alpha amylase attacks the alpha-1-4-glycosidic

bonds of starches breaking them down to the disaccharide

maltose.

Lipolytic Enzymes

1. Lipase: It partially hydrolyzes neutral fats, splitting off

one fatty acid at a time, thus forming diglycerides and

monoglycerides along with liberated free fatty acids. Its

optimum pH range is 7 to 9. This enzyme is activated

by biliary contents. It shows optimal activation when

the substrate is in emulsified form rather than in true

solution form. The emulsifying action of bile salts and

bile acids is very helpful for optimal enzymic action.

2. Lecithinase (phospholipase): Phospholipases A and

B act in succession. Both of these remove fatty acids,

the end products formed from lecithin and cephalin

are glyceryl phosphoryl choline, glyceryl phosphoryl

ethanolamine and glyceryl phosphoryl serine.

Acute Pancreatitis

Acute pancreatic necrosis (acute hemorrhagic pancreatitis)

has over 50% mortality rate. It is known to be related to

gallstones, alcoholism, trauma, infection (mumps), renal

transplantation, various metabolic disorders, e.g. hyperlipidemia, uremia and hyperparathyroidism.

Serum amylase estimation has been widely used in

the diagnosis of acute pancreatitis. Serum amylase activity

rises within hours following an episode. Values over 5 times

the upper limit of normal are suggestive of the diagnosis.

Values may return to normal within 5 days following a mild

edematous attack. Persisting elevated values longer than

this suggest continuing necrosis or possible pseudocyst

formation. The urine amylase activity rises promptly, often

within several hours of the rise in serum activity. Values

over 1,000 units per hour (in urine) or higher are seen,

almost exclusively in patients with acute pancreatitis.

Amylase activity in blood (or in peritoneal fluid in

certain conditions) may be raised to 1,000 Somogyi units

in various (nonpancreatic) disorders as: (i) intestinal

obstruction, strangulation, or perforation, (ii) following

upper abdominal surgery, (iii) ruptured ectopic pregnancy,

(iv) mumps, (v) renal insufficiency, and (vi) following

morphine administration. Values over 5,000 units suggest

a diagnosis of acute pancreatic necrosis.

Chronic Pancreatitis (Cirrhosis of Pancreas)

There is variable degree of fibrosis and atrophy in the

pancreatic parenchyma. Diagnosis as in the case of acute

pancreatitis, depends in part on determination of amylase

activity in serum and urine.

Carcinoma of Pancreas

Serum amylase may be elevated but is of little diagnostic

importance.

Lipase

This is an esterase acting on ester linkages in triglycerides.

Bile salts and calcium enhance its activity. Lipase occurs

predominantly in the pancreas, but small amounts are

produced in the gastric and small bowel mucosa.

Principle (Lipase estimation in serum)

The classic method of serum lipase determination is that

of Cherry and Crandall using olive as substrate, overnight

incubation (24 hours), and titration of liberated fatty

acids with sodium hydroxide, using phenolphthalein

as indicator. Normal range of values is up to 1.5 units in

serum.

The method given above takes a long time and if the

report is to be given on emergency basis—a rapid (20 minute

incubation) specific turbidimetric method is available.

The disadvantage of this method is spuriously high results

432 Concise Book of Medical Laboratory Technology: Methods and Interpretations obtained in the presence of jaundice. By this method,

values above 10 units are doubtful, above 19 units definitely

abnormal.

Interpretation

Acute pancreatitis: Serum lipase activity rises slower than

that of amylase, sometimes as late as 24 to 48 hours after

onset, often peaking on the fourth day. It may remain

elevated longer than the serum amylase. Eventhough,

it is less sensitive than the serum amylase, it provides

confirmatory evidence for the diagnosis when positive.

Elevation in patients with mumps strongly suggests

significant pancreatic as well as salivary gland involvement

by the disease.

Chronic pancreatitis: Serum lipase estimation is of relatively little value in the diagnosis of chronic pancreatitis.

Pancreatic carcinoma: Serum lipase is elevated more

often in patients with pancreatic carcinoma than is serum

amylase, although not with sufficient frequency to make it

of much value diagnostically.

Secretin Test

A double lumen tube, providing for separate aspiration

of gastric and duodenal contents, is passed into the

duodenum, using fluoroscopic guidance and maintaining

constant aspiration of gastric contents. Duodenal contents

are aspirated until clear. The patient is then given IV one unit

of secretin per kg of body weight, and pancreatic secretion

entering the duodenum is collected for 80 minutes. The

aspirate is examined for volume, bicarbonate content, and

amylase activity.

The test is not employed for the diagnosis of acute

pancreatic necrosis (it would be hazardous). Patients

with chronic pancreatitis are unable to secrete juice

of high bicarbonate content (less than 90 mEq/L). As

in the case of chronic pancreatitis, this test may assist

in diagnosis of pancreatic carcinoma tumors of head

of pancreas tend to depress the overall volume flow

(lower limit of normal—2 mL per kg body weight per

80 minutes). In carcinoma body of pancreas half the

patients may show normal volume, carcinoma of tail

does not affect the volume.

Patients with ductal obstructive lesion may exhibit

elevation of serum amylase during and following the

test, normally there is no elevation of serum amylase

activity. The pattern of increased volume with decreased

bicarbonate and normal amylase has been associated

with hemochromatosis. Rarely, an increase in the amylase

with normal bicarbonate concentration and volume flow

has been noted in patients with nutritional and metabolic

pancreatic fibrosis as well as in pancreatitis associated with

inflammatory disease of the intestines. In some patients of

pancreatic ductal obstruction, levels may rise.

Tumors of the head of the pancreas associated

with jaundice must be differentiated from nonsurgical

cholestatic liver disease, from carcinoma, obstructing

stone, or other obstructing pathologic lesions of the

common bile duct, and from ampullary carcinoma.

Duodenal aspirate containing cholesterol crystals or

calcium bilirubinate pigment and pus, especially when

associated with a normal secretin test, suggest gallstone

etiology. However, a duodenal aspirate containing calcium

bilirubinate pigment is not specific for cholelithiasis.

Unremittent jaundice, alcoholic duodenal fluid and stools,

consistently negative urine urobilinogen tests, and less

than 5 mg fecal urobilinogen per 24 hours, associated

with a normal secretin test, suggest carcinoma of the

common bile duct or gallbladder. Intermittent jaundice

and presence of blood in the aspirate suggest carcinoma of

the duodenal papilla, especially when associated with an

abnormal secretin test. Cytologic examination of aspirate

may be helpful in the diagnosis of carcinoma, as are the

results of enzyme and volume outputs.

Other Laboratory Tests in Acute Pancreatitis

¾ Leukocytosis in patients with acute pancreatitis (up to

30,000/mm3

).

¾ Hemoconcentration, so raised hemoglobin.

¾ Serum levels of lecithinase A, trypsin and deoxyribonuclease activity are also elevated.

¾ A falling serum calcium points to the more serious form

of pancreatitis as does turbidity of serum.

¾ In alcohol-related pancreatitis serum bilirubin may rise.

¾ Transient hyperglycemia may also occur.

Miscellaneous Tests for Chronic Pancreatitis

Various tests for malabsorption can be done

¾ Serum carotenoid level

¾ Glucose tolerance test

¾ Three-day fecal fat determination

¾ Gross and microscopic examination of stool

• 131I triolein test

• D-xylose test.

SWEAT ELECTROLYTES PILOCARPINE

IONTOPHORESIS

Pilocarpine is iontophoresed into the skin to stimulate

locally increased sweat gland secretion. The resulting

Examination of Gastrointestinal Contents 433

sweat is absorbed by filter paper, diluted with distilled

water and analyzed for sodium and chloride contents. The

method is painless and reliable. Total body sweating is

hazardous in cystic fibrosis patients.

Diagnostic Application of Sweat Testing

Fibrocystic disease of pancreas (Mucoviscidosis)

This is a familial, Mendelian recessive disease characterized by abnormal secretion by the various exocrine

glands of the body, including pancreas, salivary glands,

peritracheal, peribronchial and peribronchiolar glands,

lacrimal glands, sweat glands, mucosal glands of small

intestine and even the bile ducts.

Laboratory diagnosis depends largely upon demonstration of increased sodium and chloride in the sweat,

found in almost 99% of patients. Screening tests for sweat

chloride have also been used and depend upon hand

imprints on silver nitrate containing agar or paper. The

sweat chloride precipitates with silver, and the intensity

of the print is roughly proportional to the sweat chloride

precipitation. However, chemical estimation of sweat

chloride is more accurate.

In adult males values of sweat chloride up to 70 mEq/L

and in females up to 65 mEq/L are normal.

Normal values in children

Chloride Sodium

Below 50 mEq/L: Normal Below 70 mEq/L: Normal

50-60 mEq/L: Equivocal 70-90 mEq/L: Equivocal

Over 60 mEq/L: Abnormal Over 90 mEq/L: Abnormal

17

 

 or depression.

The 1 hour morning aspiration has replaced the

cumbersome and inherently less precise 12 hours nocturnal aspiration.

Method

1. Following a 12 hours overnight fast, the patient is

intubated. Water may be taken until 8 hours prior to

intubation.

2. The residual volume of gastric secretion is measured

and qualitatively examined.

3. Continuous aspiration is begun, preferably manually

with a syringe. Segregate the aspirate into 15 minute

samples. Usually, the first 1 or 2 samples are discarded

to allow the patient to adjust to the intubation

procedure. Subsequent to this adjustment period, four

15 minutes samples are taken.

4. If the basal secretion study is to be followed by the

augmented histamine test, a suitable dose of antihistamine be given parenterally 30 minutes before

completing the collection of basal secretion.

5. For each 15 minutes sample, the volume, pH, and

titrable acidity are measured and the acid output

calculated. The sum of the acid outputs in the 4 samples,

expressed in milliequivalents, represents the 1 hour

basal acid output.

Interpretation

The mean basal acid output reported for normal males

ranges from 1.3 to 4.0 mEq/h. Lower values occur in females

and with ageing. Somewhat lower values are reported in

most large series for gastric carcinoma and benign gastric

428 Concise Book of Medical Laboratory Technology: Methods and Interpretations ulcer and distinctively higher values for duodenal ulcer

or jejunal ulcer following partial gastrectomy with gastrojejunostomy. Extremely high acid output is present in

patients with the Zollinger-Ellison syndrome.

Augmented Histamine Test (AHT)

A dose of 0.04 mg per kg body weight is the optimum

dosage that can be given, and any further increase in

dosage does not increase the gastric acid output. All

parietal cells capable of acid secretion are stimulated by

histamine (functioning parietal cell mass). The AHT or the

analogous Histalog test are now established as definitive

tests for the diagnosis of anacidity.

The side effects of histamine are overcome by previous

administration of antihistamine. A history of bronchial

asthma or urticaria, the presence of severe cardiac,

pulmonary or renal disease and paroxysmal hypertension

or other possible signs and symptoms of pheochromocytoma are contraindications to the performance of this

test.

Method

1. Following a 12 hours fast, basal secretion is collected

for 1 hour as previously described.

2. Thirty minutes before completion of the basal

secretion collection, a suitable dose of antihistamine

is given IM, e.g. 10 mg chlorpheniramine maleate or

50 mg diphenhydramine hydrochloride.

3. After the conclusion of the basal secretion study,

histamine acid phosphate is administered subcutaneously in a dose of 0.04 mg per kg body weight.

4. Gastric contents are then collected in 15 minute

samples for 1 hour.

5. The volume, pH and titrable acidity are measured for

each sample and the acid output is calculated. From

these, the 1 hour or maximal acid output in mEq is

computed.

Interpretation

The maximum rate of acid secretion is characteristically

attained within 15 minutes after histamine injection and is

maintained for approximately 30 minutes. By 60 minutes

after histamine injection, acid secretion usually falls to the

basal level. The maximum output, representing the sum of

the acid.

The upper limit of normal is 30 mEq HCl secreted in the

30 minutes period between 15 and 45 minutes after the

histamine injection. Values higher than the stated upper

normal limit are usually found in duodenal ulcer and

Zollinger-Ellison syndrome. Anacidity in the augmented

histamine test is most commonly found in adults with

pernicious anemia or gastric carcinoma, it has also been

reported in other conditions, e.g. hypochromic anemia,

rheumatoid arthritis, steatorrhea, aplastic anemia,

myxedema, nutritional megaloblastic anemia and the

asymptomatic relatives of patients with pernicious anemia.

The basal and AHT are used as determining factors

for gastrectomy or vagotomy. It has been suggested that

an increased functioning parietal cell mass evidenced by

an elevated maximal acid output indicates the need for

gastric resection. Whereas, raised basal secretion with

normal or only slightly elevated maximal secretion is taken

as an indication for vagotomy.

Histamine Infusion Test

The use of a slow IV infusion of histamine allows

measurement of acid output in a sustained steady state.

Advantages

1. It obviates the need for doing both basal and

augmented histamine tests.

2. The greater acid output achieved in the sustained

steady state facilitates the detection of low levels of

acid output.

3. This is a highly reproducible test.

4. The slow histamine infusion has lesser side effects.

Method

1. The patient is intubated following a 12 hours overnight

fast.

2. A basal hour collection is obtained.

3. Thirty minutes before completion of the basal hour, a

suitable dose of antihistamine is given intramuscularly.

4. After completion of the basal hour, an IV infusion

of histamine in physiologic saline is begun and the

dose rate is adjusted to deliver 0.04 mg of histamine

phosphate per kg body weight per hour.

5. The infusion is continued until four 15 minute steady

state samples have been collected. The initiation of

the steady state is evident from the plateau reached

in volume output and usually requires about 30 to 45

minutes to obtain after the start of the infusion.

6. Each sample of the basal hour and steady state is

analyzed for volume, pH and titrable acidity.

Interpretation

The normal values of acid output in mEq/hour for males is

16 to 32 and for females 18 to 25. The values are markedly

higher in duodenal ulcer patients.

Histalog Test

Histalog (3 β-aminoethyl pyrazole dihydrochloride,

Betazole), an analog of histamine can be used instead of

histamine.

Examination of Gastrointestinal Contents 429

Advantage: Lesser side effects and obviation of the need to

give antihistamine.

The augmented Histalog dosage is 1.7 mg/kg given IM.

The test is similar as AHT except that: (i) no antihistamine

is needed, and (ii) eight instead of four 15 minute postHistalog samples are collected.

The peak acid secretion in Histalog test is reached in the

second to fifth 15-minute period. The peak secretory rate

may last for 45 to 90 minutes.

Insulin Hypoglycemia Test

Acid secretion is stimulated by hypoglycemia caused by

insulin administration. The major stimulus is transmitted

via vagus nerve and can be removed by vagotomy.

Hypoglycemic response—for about 30 minutes after

insulin injection there is a slight depression of gastric

secretion.

The predominant effect during the remainder of the

first 2 hours consists of marked enhancement of gastric

secretion.

The final effect is manifested after 2 hours and is also

stimulatory to gastric secretion (the second phase is via

the vagus and the third is humoral via the adrenocortical

hormones—hence the second but not the third stage can

be abolished by vagotomy).

Method

1. Following a 12 hours overnight fast the patient is

intubated. Two hours basal secretion is obtained in

15 minutes samples.

2. Blood samples for glucose estimation are obtained

upon completion of the basal secretion study and at

30, 60 and 90 minutes after insulin injection.

3. Insulin is given IV either at a fixed dosage of 15 or 20

units or at a calculated dosage of 0.20 units per kg of

body weight (keep a 50 mL syringe filled with 50%-w/vglucose solution readily available to counteract any

serious hypoglycemic effects).

4. Gastric secretion is collected in 15 minute samples for

2 hours after insulin.

5. For each basal and postinsulin gastric sample, the

volume and titrable acidity are determined, and the

acid output is computed.

Interpretation

This test is valid only if the blood glucose falls below

50  mg% at some point of the test, which will usually be

30 minutes after insulin administration. Validity of the test

also depends upon the capability of the stomach to secrete

hydrochloric acid. Hence, if no acid is present in either

the basal or postinsulin periods, it is necessary to perform

an augmented histamine test in an attempt to evoke acid

secretion. If the stomach is truely anacidic, no conclusion

can be drawn regarding the completeness of vagotomy, but

the question of simple peptic ulceration is then effectively

excluded.

The patient can be considered completely vagotomized

if the acid output in the greater of the two postinsulin

hours is less than the greater of the two basal hours.

Incomplete vagotomy is likely if the acid output in the 2

hour postinsulin period exceeds that of the 2 hour basal

period by more than 0.5 mEq. Incomplete vagotomy is

also suggested by an acid output of greater than 2 mEq

in either basal hour. In incomplete vagotomy if acid output is elevated in the first postinsulin hour—the prognosis

is bad in the sense that recurrence may occur; whereas,

elevation in the second hour is less likely to be followed by

a recurrence.

Gastrin Secretory Test

One mg of gastrin (prepared from gastric antrum of Swine)

per kg of body weight can be given subcutaneously or else

a single 50 g IV injection can be given. Most subjects will

show a maximum output beginning about 20 minutes

after gastrin injection and will maintain this level of acid

output for 20 to 40 minutes. The response is quite rapid

with IV administration, with peak levels occurring in 5 to

10 minutes.

Pentagastrin (a synthetic pentapeptide with gastrin

nucleus) can be used instead of gastrin, the results are

reproducible and without the side effects of histamine.

Miscellaneous Investigations

Mycobacterial culture: Individuals having pulmonary

tuberculosis but cannot produce sputum or in children

who cannot effectively expectorate, this method of

aspirating and culturing the gastric contents is quite useful.

It is essential that the gastric contents be collected in the

early morning prior to eating or drinking and preferably

immediately upon awakening before increased gastric

motor activity has largely emptied its contents. The sample

withdrawn should be immediately submitted for culturing.

Exfoliative cytology: For diagnosing gastric carcinomas—

gastric cytology, gastroscopy and roentgenography—

can be used, but the most discriminating information is

provided by exfoliative cytology (chymotrypsin can be

used to facilitate the exfoliation of cells by liquefying the

mucus coating). Diagnosis rate is almost 90%.

430 Concise Book of Medical Laboratory Technology: Methods and Interpretations EXAMINATION OF DUODENAL CONTENTS

Duodenal Drainage

Indications

1. For diagnosis of liver or biliary tract disease. Drainage

may be done to help diagnose exacerbations of chronic

infections early so that they can be controlled.

2. For other diagnostic purposes relating to parasites,

pancreatic enzyme, etc.

3. For therapeutic drainage in cholangitis or biliary

obstruction.

Method for Diagnostic Drainage

1. Give nothing orally after midnight.

2. In the morning intubate (Rehfuss or Levintube)

to a length of 50 cm (29 inches). Withdraw gastric

specimen.

3. With the patient erect or lying on his right side before

the fluoroscope, feed and massage tube into middle

third of the duodenum. Now aspirate duodenal

contents for 5–30 minutes and label “A”, this evacuation

specimen is of little value for bile study.

4. Slowly inject 50 mL of warm 33% magnesium sulfate

through the tube to relax sphincter of Oddi. Clamp

tube for 5 minutes then drain for 30 minutes and label

“B”. Gallbladder bile is first dark, then lighter. If no “B”

bile is obtained, inject another 50 mL of magnesium

sulfate. If still unsuccessful, inject 30 mL of olive oil.

5. During the final period of 30 minutes, try to collect

yellow hepatic bile. Label it “C”.

Examination for Diagnosis

1. Note density, color, and flocculi in all three specimens.

Test for bile, blood, reaction, and ferments as necessary.

2. Microscopy: This is important in detecting early

cholelithiasis (gallsand). Note pus cells, bacteria,

cellular elements and crystals.

3. Giardia or other parasites may be present.

4. Culture for bacteria, especially typhoid bacilli.

Interpretation

1. Absence of dark “B” bile indicates loss of gallbladder

function. No bile may appear in common duct

obstruction.

2. In cholelithiasis, many cholesterol and calcium

bilirubinate crystals appear in “B” and “C” bile. The

cholesterol crystals may be perfect or atypical or

may be mixed with cellular detritus. The calcium

bilirubinate comes as yellow or reddish particles in

the size of a pinhead.

3. In biliary tract inflammation, there is much yellow

cellular and bacterial materials in “B” and “C” bile.

4. Blood may be grossly visible in advanced carcinoma.

COMPOSITION OF BILE

Gross and Chemical Characteristics

a. Volume per 24 hours: 700–1000 mL

b. Specific gravity: Hepatic duct—1.01, gallbladder

—1.026 to 1.032.

c. Total Solids

 Hepatic duct Gallbladder

 (g%) (g%)

• Bile salts 1.8 8.7

• Fatty acids

 and lipids 0.24 1.8

• Cholesterol 0.16 0.87

d. pH : Hepatic duct, 7.5 (6.2–8.5);

 gallbladder, 6.0 (5.6–8.0)

e. Sodium : 134–156 mEq/L

f. Potassium : 3.9–6.3 mEq/L

g. Chloride : 83–110 mEq/L

h. Bicarbonate : 38 mEq/L.

PANCREATIC FUNCTION TESTS

Composition of Pancreatic Juice

Obtain specimen by duodenal drainage, it is mixed with

bile. The flow of pancreatic juice is stimulated by an

injection of secretin. Secretin is a hormone normally

produced by upper intestinal mucosa in response to

the presence of acid. The flow of pancreatic juice begins

5 minutes after a meal, is at its height in 2–3 hours, lasts

6–8 hours in all.

Gross and Chemical Characteristics of Pancreatic Juice

a. Volume per 24 hours: 500–800 mL.

b. Specific gravity: 1.007.

c. Total solids: 1.5–2.5 g%.

d. Alkalinity: pH is 7.0–8.2; 10 mL of pancreatic juice =

10–13 mL of 0.1 N NaOH and is more effective than

bile or succus entericus in neutralizing acidic gastric

juice.

e. Bicarbonate: 70–100 mEq/L.

f. Sodium: 100–150 mEq/L.

g. Potassium: 2–8 mEq/L.

h. Chlorides: 50–95 mEq/L.

Examination of Gastrointestinal Contents 431

Digestive Enzymes

Proteolytic Enzymes

1. Trypsin is a pancreatic protease. There are 100-200

units/L. It is much more active than pepsin. The inactive

trypsinogen secreted is activated by enterokinase or by

trypsin itself. Trypsin hydrolyzes proteins at peptide

bonds.

2. Chymotrypsin: Two forms, A and B are secreted and

are activated by trypsin. Its action is like that of trypsin.

3. Collagenase: It digests collagen and is the one that

initiates tissue destruction in necrotizing pancreatitis.