Page 324 - Textbook of Pathology, 6th Edition

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replaced the traditional microbiologic assays for vitamin If the 24-hour urinary excretion of ‘hot’ B is now
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B and folate. Traditional tests are briefly described below. normal, the low value in first stage of the test was due to
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IF deficiency (i.e. pernicious anaemia).
TESTS FOR VITAMIN B 12 DEFICIENCY. The normal
range of vitamin B in serum is 280-1000 pg/ml. Values Patients with pernicious anaemia have abnormal test
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less than 100 pg/ml indicate clinically deficient stage. even after treatment with vitamin B due to IF deficiency.
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Traditional tests employed to establish vitamin B 12 However, abnormal 24-hour urinary excretion of ‘hot’ B 12
deficiency are serum vitamin B assay, Schilling (24-hour is further investigated in stage III for a cause in intestinal
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urinary excretion) test and serum enzyme levels. malabsorption of ‘hot’ B’ .
Stage III: Test for malabsorption of vitamin B . Some
1. SERUM VITAMIN B ASSAY. Assay of vitamin B in 12
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blood can be done by 2 methods—microbiological assay patients absorb vitamin B in water as was stipulated in
and radioassay. the original Schilling test. Modified Schilling test employs
the use of protein-bound vitamin B . In conditions
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i) Microbiological assay. In this test, the serum sample to causing malabsorption, the test is repeated after a course
be assayed is added to a medium containing all other of treatment with antibiotics or anti-inflammatory drugs.
essential growth factors required for a vitamin
B -dependent microorganism. The medium along with 3. SERUM ENZYME LEVELS. Besides Schilling test,
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microorganism is incubated and the amount of vitamin another way of distinguishing whether megaloblastic
B is determined turbimetrically which is then compared anaemia is due to cobalamine or folate is by serum
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SECTION II
with the growth produced by a known amount of vitamin determination of methylmalonic acid and homocysteine
B . Several organisms have been used for this test such by sophisticated enzymatic assays. Both are elevated in
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cobalamine deficiency, while in folate deficiency there is
as Euglena gracilis, Lactobacillus leichmannii, Escherichia coli only elevation of homocysteine and not of methylmalonic
and Ochromonas malhamensis. E. gracilis is, however, consi-
dered more sensitive and accurate. The addition of anti- acid.
biotics to the test interferes with the growth and yields TESTS FOR FOLATE DEFICIENCY. The normal range
false low result. of serum folate is 6-18 ng/ml. Values of 4 ng/ml or less
are generally considered to be diagnostic of folate
ii) Radioassay. Assays of serum B by radioisotope
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dilution (RID) and radioimmunoassay (RIA) have been deficiency. Measurement of formiminoglutamic acid
(FIGLU) urinary excretion after histidine load was used
developed. These tests are more sensitive and have the formerly for assessing folate status but it is less specific
advantage over microbiologic assays in that they are and less sensitive than the serum assays. Currently, there
simpler and more rapid, and the results are unaffected by are 3 tests used to detect folate deficiency—urinary
antibiotics and other drugs which may affect the living excretion of FIGLU, serum and red cell folate assay.
organisms.
1. URINARY EXCRETION OF FIGLU. Folic acid is
2. SCHILLING TEST (24 HOUR URINARY EXCRETION required for conversion of formiminoglutamic acid
TEST). Schilling test is done to detect vitamin B 12 (FIGLU) to glutamic acid in the catabolism of histidine.
deficiency as well as to distinguish and detect lack of IF Thus, on oral administration of histidine, urinary excretion
and malabsorption syndrome. The results of test also of FIGLU is increased if folate deficiency is present.
depend upon good renal function and proper urinary 2. SERUM FOLATE ASSAY. The folate in serum can be
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collection. Radioisotope used for labeling B is either Co estimated by 2 methods—microbiological assay and
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or Co. The test is performed in 3 stages as under:
radioassay.
Haematology and Lymphoreticular Tissues
Stage I: Without IF. The patient after an overnight fasting i) Microbiological assay. This test is based on the principle
is administered oral dose of 1 μg of radioactively labelled that the serum folate acid activity is mainly due to the
vitamin B (‘hot’ B ) in 200 ml of water. At the same presence of a folic acid co-enzyme, 5-methyl THF, and
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time, 1 mg of unlabelled vitamin B (‘cold’ B ) is given that this compound is required for growth of the
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by intramuscular route; this ‘cold’ B will saturate the microorganism, Lactobacillus casei. The growth of L. casei
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serum as well as the tissue binding sites. The patient is is inhibited by addition of antibiotics.
kept fasting for a further period of 2 hours, following ii) Radioassay. The principle and method of radioassay
which urinary excretion of B is estimated: by radioisotope dilution (RID) test are similar to that for
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In normal individuals, 24-hour urinary excretion is serum B assay. The test employs labelled pteroyl-
>10% of the oral dose of ‘hot’ B . 12
12 glutamic acid or methyl-THF. Commercial kits are
Patients with IF deficiency excrete lower quantity of available which permit simultaneous assay of both
‘hot’ B which is further confirmed by repeating the test vitamin B and folate.
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as in stage II given below. 12
3. RED CELL FOLATE ASSAY. Red cells contain 20-50
Stage II: With IF. If the 24-hour urinary excretion of ‘hot’ times more folate than the serum; thus red cell folate assay
B is low, the test is repeated using the same procedure is more reliable indicator of tissue stores of folate than
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as in stage I but in addition high oral dose of IF is serum folate assay. Microbiological radioassay and
administered along with ‘hot’ B . protein-binding assay methods can be used for estimation
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