CA1039185A - Hypothyroid serum control - Google Patents
Hypothyroid serum controlInfo
- Publication number
- CA1039185A CA1039185A CA227,609A CA227609A CA1039185A CA 1039185 A CA1039185 A CA 1039185A CA 227609 A CA227609 A CA 227609A CA 1039185 A CA1039185 A CA 1039185A
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- CA
- Canada
- Prior art keywords
- serum
- hypothyroid
- uptake
- supernatant
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/106664—Blood serum or blood plasma standard or control
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
- Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
ABSTRACT
The present invention describes the preparation of a hypothyroid serum control using normal serum and serum containing elevated levels of thyroxine binding globulin. The control serum exhibits hypothyroid values in clinical tests that measure triiodothyronine uptake and thyroxine concentration.
The present invention describes the preparation of a hypothyroid serum control using normal serum and serum containing elevated levels of thyroxine binding globulin. The control serum exhibits hypothyroid values in clinical tests that measure triiodothyronine uptake and thyroxine concentration.
Description
1()35~5 To diagnose thyroid disfunction and evaluate thyroid therapy, clinicians rely on tests which measure a patient's serum triiodithyronine (T3) uptake level as well as tests which do quantitate the thyroxine (T4) concentration. T3 uptake levels are determined in serum using T3 uptake tests (e.g., Triosorb*); serum T4 levels are quantitated using tests which measure total serum T4 concentration (e.g. T4 radioimmunoassay, Tetrasorb*).
Tests measuring total serum T4 are dependent upon the actual thyroid hormone concentration present in the serum, whereas tests which measure T3 uptake depend upon the thyroxine-binding protein (TBP) concentration in the serum as well as the concentration of thyroid hormone. Both tests are important in forming a clear understanding of the patient's thyroid stateO
Circulating T3 and T4 are bound to several constituents of the blood of which the thyroxine binding globulin (TBG~ fraction contains the major binding sites which are fixed in number. The binding of T3 and T4 on the TBG molecule is one of competitive protein binding, that is, unbound T4 will replace T3 and T4 already on the molecule.
As there is only 1-2 mg of TBG/100 ml normal serum, the binding sites, equivalent to approximately 20 micrograms of T4 per 100 ml of normal serum, are readily saturated by a small increase of T4 concentration.
In hyperthyroidism, the binding sites on the TBG molecules are nearly saturated with T3 and T4; in hypothyroidism, the binding sites are highly unsaturated, resulting in an increased ability for T3 uptake by the TBG molecule in the serum.
The Triosorb* assay and other similar tests for T3 uptake operate on the principle of competitive protein binding. Serum T3 and T4 are primarily bound to the binding sites on the TBG molecule. The number of unoccupied binding sites is determined by the addition of radioactive T3 (T3*) to serum in the presence of an adsorbing agent. When T3* is added to serum, any excess not bound to the binding sites of the TBG molecule in the serum will be ad-sorbed onto the added agentO Resin sponges are the adsorbing agents for the *Trade Mark _ _ ~r Al ~ 03~85 Triosorb test. For example, in hyperthyroidism, most of the TBG binding sites are occupied by T3 and T4 and thus the added T3* will not be taken up by the endogenous TBG molecules, but will be taken up by the test adsorbent. In hypothyroidism, the reverse is true. Thus~ the amount of radioactive T3 bound by the adsorbent directly reflects the thyroid state of the patient.
The tetrasorb assay for T4 is also based upon the principle of competitlve protein binding. To determine the T4 concentration, T4 is extracted from the serum releasing it from its binding protein, TBG. The serum extract is then added to a solution containing a limited quantity of exogenous TBG to which is bound radioactive T4 (T4*). A displacement reaction occurs in which the T4 in the extract displaces the T4* from the exogenous TBG. This dis-placed T4~ is then adsorbed onto the resin sponge used as the adsorbing agent.
As more T4 is added, more T4* is displaced from the TBG. The amount Or T4*
displaced from the TBG is therefore directly proportional to the amount of T4 present in the serum extract. In hyperthyroidism there is more T4 available in the serum extract to displace T4~ from exogenous TBG than in euthyroidism or hypothyroidism.
When normal levels of TBG are present in serum, these types of thyroid function tests reflect the actual state of the patient. When TBG and T4 levels are elevated, as in pregnancy or following estrogen ingestion in the form of oral contraceptives, T3 uptake tests indicate the patient to be hypo-thyroid. However, tests measuring total T4 indicate an euthyroid or sometimes a hyperthyroid condition. Si~lply stated, pregnancy or estrogen ingestion re-sults in an increase in the number of TBG molecules causing an increase in hormone binding sites with a concurrent rise in thyroid hormone levels.
Thyroid function tests carried out on these sera at this time show an increased T3 uptake, indicative of hypothyroid function and an increased T4 level indi-cative of hyperthyroid function.
This invention seeks to provide a control serum prepared from these types of sera and normal serum that will serve as a hypothyroid control serum for T4 tests as well as T3 uptake tests. By normal serum Applicants mean serum havi~ norm~l T3 and T4 values and which may be human, beef, sheep, goat, or other animal serum. Only horse serum has been found to lack utility.
In à first aspect this invention seeks to provide a method for ob-taining a hypothyroid serum control comprising the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture for about 24 hours at a temperature of about 4C;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4C;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
In a second aspect this invention seeks to provide in clinical chemis-try procedures measuring a person's serum triiodothyronine uptake level and thyroxine concentration, the improvement which comprises the use of a single control serum standard as a hypothyroid reference control serum for both the triiodothyronine uptake and thyroxine concentration tests, in which the hypo-thyroid reference control serum is prepared by the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture at a temperature of about 4c;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4c;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
A serum is hyperthyroid by the tetrasorb and triosorb tests if the percentage of T3 uptake is greater than 35, and the T4 concentration is above 14.5 mcg per 100 ml of serum. A serum is euthyroid if the percentage of T3 up-take is 25 to 35 and the T4 concentration is 5.3-14.5 mcg per 100 ml of serum.
Hypothyroid serum has a T3 uptake of below 25 percent and less than 5 mcg of T4 per 100 mls of serum.
~ _ 3 _ V.
10391~35 Removing T3 and T4 from normal serum or serum containing elevated TBG levels, such as by the methods outlined below, results in a serum judged hypothyroid both by methods which measure T4 concentration and T3 uptake.
- 3a -~B
~ g~fl5 Ingredients Quantity for 990 m1 ~420 vials) lo Fresh, Normal Serum 2700 ml 20 Norit A*, Neutral Decolorizing480 g Carbon pharmaceutical grade, ~Amend Drug CoO, Irvington, N.J.) 3. Water, Purified 560 ml Method 1. Add Item #2 to 2400 ml of Item #1 in a 4 liter Erlenmeyer flask (about 20% carbon weight to volume of serum).
Tests measuring total serum T4 are dependent upon the actual thyroid hormone concentration present in the serum, whereas tests which measure T3 uptake depend upon the thyroxine-binding protein (TBP) concentration in the serum as well as the concentration of thyroid hormone. Both tests are important in forming a clear understanding of the patient's thyroid stateO
Circulating T3 and T4 are bound to several constituents of the blood of which the thyroxine binding globulin (TBG~ fraction contains the major binding sites which are fixed in number. The binding of T3 and T4 on the TBG molecule is one of competitive protein binding, that is, unbound T4 will replace T3 and T4 already on the molecule.
As there is only 1-2 mg of TBG/100 ml normal serum, the binding sites, equivalent to approximately 20 micrograms of T4 per 100 ml of normal serum, are readily saturated by a small increase of T4 concentration.
In hyperthyroidism, the binding sites on the TBG molecules are nearly saturated with T3 and T4; in hypothyroidism, the binding sites are highly unsaturated, resulting in an increased ability for T3 uptake by the TBG molecule in the serum.
The Triosorb* assay and other similar tests for T3 uptake operate on the principle of competitive protein binding. Serum T3 and T4 are primarily bound to the binding sites on the TBG molecule. The number of unoccupied binding sites is determined by the addition of radioactive T3 (T3*) to serum in the presence of an adsorbing agent. When T3* is added to serum, any excess not bound to the binding sites of the TBG molecule in the serum will be ad-sorbed onto the added agentO Resin sponges are the adsorbing agents for the *Trade Mark _ _ ~r Al ~ 03~85 Triosorb test. For example, in hyperthyroidism, most of the TBG binding sites are occupied by T3 and T4 and thus the added T3* will not be taken up by the endogenous TBG molecules, but will be taken up by the test adsorbent. In hypothyroidism, the reverse is true. Thus~ the amount of radioactive T3 bound by the adsorbent directly reflects the thyroid state of the patient.
The tetrasorb assay for T4 is also based upon the principle of competitlve protein binding. To determine the T4 concentration, T4 is extracted from the serum releasing it from its binding protein, TBG. The serum extract is then added to a solution containing a limited quantity of exogenous TBG to which is bound radioactive T4 (T4*). A displacement reaction occurs in which the T4 in the extract displaces the T4* from the exogenous TBG. This dis-placed T4~ is then adsorbed onto the resin sponge used as the adsorbing agent.
As more T4 is added, more T4* is displaced from the TBG. The amount Or T4*
displaced from the TBG is therefore directly proportional to the amount of T4 present in the serum extract. In hyperthyroidism there is more T4 available in the serum extract to displace T4~ from exogenous TBG than in euthyroidism or hypothyroidism.
When normal levels of TBG are present in serum, these types of thyroid function tests reflect the actual state of the patient. When TBG and T4 levels are elevated, as in pregnancy or following estrogen ingestion in the form of oral contraceptives, T3 uptake tests indicate the patient to be hypo-thyroid. However, tests measuring total T4 indicate an euthyroid or sometimes a hyperthyroid condition. Si~lply stated, pregnancy or estrogen ingestion re-sults in an increase in the number of TBG molecules causing an increase in hormone binding sites with a concurrent rise in thyroid hormone levels.
Thyroid function tests carried out on these sera at this time show an increased T3 uptake, indicative of hypothyroid function and an increased T4 level indi-cative of hyperthyroid function.
This invention seeks to provide a control serum prepared from these types of sera and normal serum that will serve as a hypothyroid control serum for T4 tests as well as T3 uptake tests. By normal serum Applicants mean serum havi~ norm~l T3 and T4 values and which may be human, beef, sheep, goat, or other animal serum. Only horse serum has been found to lack utility.
In à first aspect this invention seeks to provide a method for ob-taining a hypothyroid serum control comprising the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture for about 24 hours at a temperature of about 4C;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4C;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
In a second aspect this invention seeks to provide in clinical chemis-try procedures measuring a person's serum triiodothyronine uptake level and thyroxine concentration, the improvement which comprises the use of a single control serum standard as a hypothyroid reference control serum for both the triiodothyronine uptake and thyroxine concentration tests, in which the hypo-thyroid reference control serum is prepared by the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture at a temperature of about 4c;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4c;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
A serum is hyperthyroid by the tetrasorb and triosorb tests if the percentage of T3 uptake is greater than 35, and the T4 concentration is above 14.5 mcg per 100 ml of serum. A serum is euthyroid if the percentage of T3 up-take is 25 to 35 and the T4 concentration is 5.3-14.5 mcg per 100 ml of serum.
Hypothyroid serum has a T3 uptake of below 25 percent and less than 5 mcg of T4 per 100 mls of serum.
~ _ 3 _ V.
10391~35 Removing T3 and T4 from normal serum or serum containing elevated TBG levels, such as by the methods outlined below, results in a serum judged hypothyroid both by methods which measure T4 concentration and T3 uptake.
- 3a -~B
~ g~fl5 Ingredients Quantity for 990 m1 ~420 vials) lo Fresh, Normal Serum 2700 ml 20 Norit A*, Neutral Decolorizing480 g Carbon pharmaceutical grade, ~Amend Drug CoO, Irvington, N.J.) 3. Water, Purified 560 ml Method 1. Add Item #2 to 2400 ml of Item #1 in a 4 liter Erlenmeyer flask (about 20% carbon weight to volume of serum).
2. Swirl gently at room temperature until the charcoal particules are dispersed in the liquid.
3. Place the mixture at 4C. Stir very gently for about 24 hoursO
4. At the end of 24 hours centrifuge mixture at high speed in a refrigerated centrifuge ~34,800 x g)O
- 5. Decant the supernatant and centrifuge it again as in step 4.
6. After the second centrifugation, again decant the supernatant and filter it by vacuum through a millipore* filter~
7. Pour the filtrate (1200 ml) into a suitable glass tray for lyophiliz-ation and lyophilize.
8. Transfer the lyophilized material to a 2 liter Erlenmeyer flask and add Item #3.
9. Let the mixture stand for 30 minutes, then aid the dissolving process by swirling gently.
10. Add 300 ml of Item #l to the serum mixture obtained in step 9.
11. Dispense 2.14 cc into a vial that will hold a 2 ml fill and lyophilize.
Vials are reconstituted with 2.0 ml deionized distilled water. This serum is used for a hypothyroid control in conjunction with all thyroid function tests, i.e., the resulting serum control should have a T3 uptake level less than 25% by the Triosorb method and a T4 concentration less than 5.3 mg/100 ml by the Tetrasorb methodO
*Trade Mark ~,3 '~
10;~9~5 Modifications in Example 1 are of course possible. Centrifugation (steps 4 and 5) may be eliminated; the millipore filter (step 6) may, of course, be an Ertel apparatus; the serum may be human or animal; the require-ment to dispense the mixtures (step 11) may, of course, be modified or done away with completely; the time of mixing (step 3) may be shortened or length-ened over a range of 3-30 hours; the percentage of carbon added may range from 5-20 percent, with a range of about 10 to about 20 percent being pre-ferred.
This procedure removes over 99% of the T3 and T4 from the starting serum, effectively producing a T3 and T4-free serum while not significantly affecting the total protein concentration, pH, or T4 binding capacity of the serum.
The following example outlines the method used to remove T3 and T4 from serum containing elevated TBG levels. Modifications of this example s;m;lar to those outlined for Example l are also possible and encompassed by this invention.
1()3~1~35 Ingredients Quantity for 900 ml (420 vials~
1. Serum containing a high thyroxine 1100 ml binding globulin concentration 2. Neutral Decolorizing Carbon, 220 g pharmaceutical grade 3. Fresh, normal serum 200 ml Method 1. Add Item 2 to Item 1 in a 2 liter Erlenmeyer flask (about 20% carbon weight to volume of serum).
2. Swirl gently at room temperature for one minute so that charcoal particles are dispersed in liquid.
3. Place mixture at 4C and stir very gently for about 24 hours.
4. At the end of 24 hours, centrifuge mixture at high speed in a refrigerated centrifuge (34,800 ~ g).
5. Decant the supernatant and centrifuge it again as in Step 4.
6. After second centrifugation, again decant the supernatant and filter it by vacuum through a sintered glass filter.
7. Add 300 ml of Item 3 to 600 ml of serum obtained from Step 6.
Swirl gently to mix.
8. Dispense 2.14 cc in a vial that will hold a 2 ml fill and lyophilize.
~ials are reconstituted with 2.0 ml deionized distilled water. This serum is used for a hypothyroid control serum in conjunction with all thyroid function tests.
The fill solution after reconstitution will contain about 7 percent protein.
The resulting serum control should have a T3 uptake level less than 25% by the Triosorb Method and a T4 concentration less than 5~31ug% by the Tetrasorb Method. The serum used in this example may be obtained, for example, from pregnant women or women on estrogen therapy.
A series of four separate experiments were carried out to show the effect on sera when treated as to Examples 1 and 2. The resultant sera were assayed by following the well recognized Triosorb and Tetrasorb protocols.
The results of these experiments (Table I) show that serum treated according to the methods of this invention, may be used as a hypothyroid control serum for tests being carried out in clinical laboratories.
\
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E~
~ ,, ~
,, ~
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a~
~ ~ O ~
a ~ 1~ ~ O
- æ CH ~ 3 Eiatat at ~1 at U~ ~ ~ ~ O O
ad a~ ~ a~
* ~ ~ * * C~ ~ *
x-- 3~ ~^* ~q m--~* ~ h ~ c~
æ ~ ~ ~ ~ æ ~ ~
o o ~ o o ~--~ o o ~ ~
æ zi ~ ~; æ ~ æ z; ~
H H at r-l ~1 a,t C~H r-l a~ ~4 ~1 ~1 ~ ~ ,S;
o o ~ o o a~ 3~o o a~ r O O a~
o o h o o h o o hrJ o h ~ ~ ~ ~ ~ ~ ~0~ ~ ~ ~ ~ ~ ~ * **
O. H H H H
The results of experiment I show that the pooled normal serum was euthyroid by both tests. After treatment the serum was hypothyroid for both T3 uptake and T4 concentration.
The results for experiment II show that the hypothyroid values may be altered upwardly when before treatment sera and after treatment sera are intermixed, as in this experiment in a 2 to 1 ratio.
Experiment III was carried out with beef sera as representative of animal sera which have been shown to have utility. As the results show, pooled normal beef serum shows T3 uptake and T4 concentration in the euthyroid range. After treatment, this sera behaves similar to human sera and is in the hypothyroid range to T3 uptake and T4 concentration. As with human serum, the T3 and T4 values may be increased by mixing various ratios of normal beef serum and the prepared hypothyroid beef serum.
Experiment IV shows the expected hypothyroid T3 uptake and euthyroid T4 concentration for pooled serum collected from pregnant women. After the treatment outlined in Example 2, however, the serum becomes hypothyroid for both T3 uptake and T4 concentration.
Vials are reconstituted with 2.0 ml deionized distilled water. This serum is used for a hypothyroid control in conjunction with all thyroid function tests, i.e., the resulting serum control should have a T3 uptake level less than 25% by the Triosorb method and a T4 concentration less than 5.3 mg/100 ml by the Tetrasorb methodO
*Trade Mark ~,3 '~
10;~9~5 Modifications in Example 1 are of course possible. Centrifugation (steps 4 and 5) may be eliminated; the millipore filter (step 6) may, of course, be an Ertel apparatus; the serum may be human or animal; the require-ment to dispense the mixtures (step 11) may, of course, be modified or done away with completely; the time of mixing (step 3) may be shortened or length-ened over a range of 3-30 hours; the percentage of carbon added may range from 5-20 percent, with a range of about 10 to about 20 percent being pre-ferred.
This procedure removes over 99% of the T3 and T4 from the starting serum, effectively producing a T3 and T4-free serum while not significantly affecting the total protein concentration, pH, or T4 binding capacity of the serum.
The following example outlines the method used to remove T3 and T4 from serum containing elevated TBG levels. Modifications of this example s;m;lar to those outlined for Example l are also possible and encompassed by this invention.
1()3~1~35 Ingredients Quantity for 900 ml (420 vials~
1. Serum containing a high thyroxine 1100 ml binding globulin concentration 2. Neutral Decolorizing Carbon, 220 g pharmaceutical grade 3. Fresh, normal serum 200 ml Method 1. Add Item 2 to Item 1 in a 2 liter Erlenmeyer flask (about 20% carbon weight to volume of serum).
2. Swirl gently at room temperature for one minute so that charcoal particles are dispersed in liquid.
3. Place mixture at 4C and stir very gently for about 24 hours.
4. At the end of 24 hours, centrifuge mixture at high speed in a refrigerated centrifuge (34,800 ~ g).
5. Decant the supernatant and centrifuge it again as in Step 4.
6. After second centrifugation, again decant the supernatant and filter it by vacuum through a sintered glass filter.
7. Add 300 ml of Item 3 to 600 ml of serum obtained from Step 6.
Swirl gently to mix.
8. Dispense 2.14 cc in a vial that will hold a 2 ml fill and lyophilize.
~ials are reconstituted with 2.0 ml deionized distilled water. This serum is used for a hypothyroid control serum in conjunction with all thyroid function tests.
The fill solution after reconstitution will contain about 7 percent protein.
The resulting serum control should have a T3 uptake level less than 25% by the Triosorb Method and a T4 concentration less than 5~31ug% by the Tetrasorb Method. The serum used in this example may be obtained, for example, from pregnant women or women on estrogen therapy.
A series of four separate experiments were carried out to show the effect on sera when treated as to Examples 1 and 2. The resultant sera were assayed by following the well recognized Triosorb and Tetrasorb protocols.
The results of these experiments (Table I) show that serum treated according to the methods of this invention, may be used as a hypothyroid control serum for tests being carried out in clinical laboratories.
\
10;~ 85 a) ~o In ~ ~~ d- O ~'7 ~ ~ O ~D O ~ ~i 0 C~J ~ O
E~
~ ,, ~
,, ~
H
a~
~ ~ O ~
a ~ 1~ ~ O
- æ CH ~ 3 Eiatat at ~1 at U~ ~ ~ ~ O O
ad a~ ~ a~
* ~ ~ * * C~ ~ *
x-- 3~ ~^* ~q m--~* ~ h ~ c~
æ ~ ~ ~ ~ æ ~ ~
o o ~ o o ~--~ o o ~ ~
æ zi ~ ~; æ ~ æ z; ~
H H at r-l ~1 a,t C~H r-l a~ ~4 ~1 ~1 ~ ~ ,S;
o o ~ o o a~ 3~o o a~ r O O a~
o o h o o h o o hrJ o h ~ ~ ~ ~ ~ ~ ~0~ ~ ~ ~ ~ ~ ~ * **
O. H H H H
The results of experiment I show that the pooled normal serum was euthyroid by both tests. After treatment the serum was hypothyroid for both T3 uptake and T4 concentration.
The results for experiment II show that the hypothyroid values may be altered upwardly when before treatment sera and after treatment sera are intermixed, as in this experiment in a 2 to 1 ratio.
Experiment III was carried out with beef sera as representative of animal sera which have been shown to have utility. As the results show, pooled normal beef serum shows T3 uptake and T4 concentration in the euthyroid range. After treatment, this sera behaves similar to human sera and is in the hypothyroid range to T3 uptake and T4 concentration. As with human serum, the T3 and T4 values may be increased by mixing various ratios of normal beef serum and the prepared hypothyroid beef serum.
Experiment IV shows the expected hypothyroid T3 uptake and euthyroid T4 concentration for pooled serum collected from pregnant women. After the treatment outlined in Example 2, however, the serum becomes hypothyroid for both T3 uptake and T4 concentration.
Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for obtaining a hypothyroid serum control comprising the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture for about 24 hours at a tempera-ture of about 4°C;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4°C;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture for about 24 hours at a tempera-ture of about 4°C;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4°C;
D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
2. The method of Claim 1 which includes adding untreated normal serum in an amount of about 10 to about 35 percent of the serum volume of step A
to the filtered supernatant of step D.
to the filtered supernatant of step D.
3. The method of Claim 1 wherein the serum is animal blood serum.
4. The method of Claim 1 wherein the serum is human blood serum.
5. The method of Claim 1 wherein the serum is beef blood serum.
6. The method of Claim 4 wherein the serum is T4-euthyroid, hypothyroid, or hyperthyroid and T3-euthyroid or hypothyroid.
7. The method of Claim 6 wherein the serum is T3-hypothyroid.
8. The method of Claim 6 wherein the serum is T4-euthyroid and T3-euthyroid.
9. A control serum judged hypothyroid by methods which measure T4 concentration and T3 uptake comprising blood serum which has been treated by the method according to Claim 1.
10. In clinical chemistry procedures measuring a person's serum triiodothyronine uptake level and thyroxine concentration, the improvement which comprises the use of a single control serum standard as a hypothyroid reference control serum for both the triiodothyronine uptake and thyroxine concentration tests, in which the hypothyroid reference control serum is prepared by the following steps:
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture at a temperature of about 4°C;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4°C, D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;
B. mixing the above mixture at a temperature of about 4°C;
C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4°C, D. filtering the resultant supernatant;
E. lyophilizing the supernatant so obtained.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US473463A US3922145A (en) | 1974-05-28 | 1974-05-28 | Hypothyroid serum control |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1039185A true CA1039185A (en) | 1978-09-26 |
Family
ID=23879629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA227,609A Expired CA1039185A (en) | 1974-05-28 | 1975-05-23 | Hypothyroid serum control |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US3922145A (en) |
| JP (1) | JPS552027B2 (en) |
| CA (1) | CA1039185A (en) |
| DE (1) | DE2517219B2 (en) |
| DK (1) | DK233575A (en) |
| FR (1) | FR2273282B1 (en) |
| GB (1) | GB1480199A (en) |
| IT (1) | IT1038419B (en) |
| MX (1) | MX3040E (en) |
| NO (1) | NO751877L (en) |
| SE (1) | SE7506104L (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4230601A (en) * | 1978-05-03 | 1980-10-28 | Eastman Kodak Company | Calibrator composition based upon dialyzed blood serum |
| EP0082191B1 (en) * | 1981-06-24 | 1987-05-20 | Cuno Incorporated | Process for preparing a zero standard serum |
| US4431741A (en) * | 1981-12-17 | 1984-02-14 | Baxter Travenol Laboratories, Inc. | Hypothyroid control serum |
| JP3363342B2 (en) * | 1997-05-14 | 2003-01-08 | 本田技研工業株式会社 | Vent device for vehicle fuel tank |
| WO2019074886A1 (en) | 2017-10-09 | 2019-04-18 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3516794A (en) * | 1964-12-14 | 1970-06-23 | Squibb & Sons Inc | Apparatus and method for determining thyroid function |
| US3743482A (en) * | 1970-12-30 | 1973-07-03 | Nuclear Med Lab | Method and apparatus for determining thyroid function |
| US3775615A (en) * | 1971-07-06 | 1973-11-27 | Nuclear Med Lab | Method of determining thyroid function |
| US3776698A (en) * | 1972-01-24 | 1973-12-04 | Nuclear Med Lab | Test for thyroid hormone |
-
1974
- 1974-05-28 US US473463A patent/US3922145A/en not_active Expired - Lifetime
-
1975
- 1975-04-18 DE DE19752517219 patent/DE2517219B2/en not_active Ceased
- 1975-04-21 MX MX000428U patent/MX3040E/en unknown
- 1975-05-14 GB GB20352/75A patent/GB1480199A/en not_active Expired
- 1975-05-23 CA CA227,609A patent/CA1039185A/en not_active Expired
- 1975-05-23 FR FR7516183A patent/FR2273282B1/fr not_active Expired
- 1975-05-26 IT IT23724/75A patent/IT1038419B/en active
- 1975-05-27 JP JP6341675A patent/JPS552027B2/ja not_active Expired
- 1975-05-27 NO NO751877A patent/NO751877L/no unknown
- 1975-05-27 DK DK233575A patent/DK233575A/en unknown
- 1975-05-28 SE SE7506104A patent/SE7506104L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| FR2273282B1 (en) | 1978-02-03 |
| DE2517219B2 (en) | 1976-11-04 |
| AU8061975A (en) | 1976-11-04 |
| GB1480199A (en) | 1977-07-20 |
| MX3040E (en) | 1980-03-04 |
| JPS552027B2 (en) | 1980-01-18 |
| US3922145A (en) | 1975-11-25 |
| IT1038419B (en) | 1979-11-20 |
| DK233575A (en) | 1975-11-29 |
| JPS513290A (en) | 1976-01-12 |
| NO751877L (en) | 1975-12-01 |
| FR2273282A1 (en) | 1975-12-26 |
| SE7506104L (en) | 1975-12-01 |
| DE2517219A1 (en) | 1975-12-11 |
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