Sunday, November 2, 2025

CERULOPLASMIN Test. lab information about test and diagnosis of diseases

______________________________________________________________________________

Ceruloplasmin Test. Lab information about test and diagnosis of diseases.

Ceruloplasmin Test – Manual of Medical Laboratory Techniques

Purpose:
To measure the amount of ceruloplasmin in human serum. Low levels are reported in Wilson’s disease and nephrotic syndrome, and it is valuable in differentiating chronic liver diseases from Wilson’s disease.

Principle:
Ceruloplasmin, a ferro-oxidase enzyme, catalyzes the oxidation of certain polyamines. Its activity on p-phenylenediamine is measured to determine the amount of ceruloplasmin in serum.

Performance Specifications:

Linearity: up to 60 mg/dL
Measurement range: 16–60 mg/dL
Sensitivity: 16 mg/dL

Primary Sample:

Use serum as the specimen.
Collect 4 mL of venous blood in a plain red vacutainer.
Allow to clot for 30 minutes, then centrifuge at 2500 rpm for 10 minutes.
Avoid hemolyzed or turbid samples.
Analyze within 3 hours of collection, or store serum at –20°C for up to 7 days.

Reagents and Consumables:

p-Phenylenediamine hydrochloride: 5 g in 1 L distilled water (purified and crystallized).
Acetic acid (1 M): 60 mL glacial acetic acid diluted to 1 L.
Sodium acetate (1 M): 136 g per liter of water.
Acetate buffer (400 mM, pH 5.5): Prepared by mixing acetic acid and sodium acetate.
Sodium azide or sodium fluoride: Preservatives (5 g/L or 20 g/L).

Instrument:
Spectrophotometer (read at 530 nm).

Procedure:

Calculation:

Reference Range:

Normal: 20 – 40 mg/dL

Critical/Alert Values:

Not applicable.

Potential Sources of Error:

Hemolyzed serum may give falsely elevated values.
Samples not analyzed promptly must be stored at –20°C to maintain accuracy.

References:
Ramakrishnan, Manual of Medical Laboratory Techniques, pp. 25–27.
King, J. Practical Clinical Enzymology, 1965.

Produce by : Dr. Yousef AL-Adbai.

ALBUMIN Test. lab information about test and diagnosis of diseases.

_______________________________________

ALBUMIN Test. Lab information about test and diagnosis of diseases.

Albumin Test – Manual of Medical Laboratory Techniques

Purpose:
Quantitative estimation of albumin in human serum by photometric method using bromocresol green (BCG) dye binding. Elevated serum albumin levels are associated with dehydration. Low serum albumin levels indicate potential malnutrition, liver diseases, kidney disorders (especially nephrotic syndrome), and rheumatoid arthritis.

Principle:
Albumin acts as a cation at pH 3.8 and selectively binds to the anionic dye bromocresol green, forming a green-colored complex. The intensity of the color, measured at 630 nm, is directly proportional to the concentration of albumin in the sample.

Performance Specifications:

Linearity: up to 6.0 g/dL
Measurement range: 0.5–6.0 g/dL
Sensitivity: 0.5 g/dL
Specificity: certain drugs (e.g., Ampicillin) may interfere with dye-binding properties. Only human albumin standards should be used.

Primary Sample:

Use serum (fasting sample recommended).
Avoid venostasis during collection to prevent hemoconcentration.
Collect 4 mL venous blood in a plain red vacutainer; allow to clot for 30 minutes.
Centrifuge at 2500 rpm for 10 minutes.
Avoid hemolyzed or turbid samples.
Analyze within 3 hours or store serum at 2–8 °C (up to 30 days).

Reagents and Equipment:

Reagent: Bromocresol Green (BCG) buffer, pH 3.68
Standard: Bovine albumin fraction V (5 g/dL)
Instrument: Semi-auto analyzer (e.g., RA 50)
Wavelength: 628 nm
Temperature: 37 °C
Incubation time: 10 minutes

Procedure:

Run reagent blank with distilled water.
Process standard, then patient samples.
Measure absorbance at 628 nm.

Calculation:

Reference Range:

Adults: 3.5 – 5.0 g/dL

Critical Values:

Below 2.0 g/dL indicates severe hypoalbuminemia.

Interpretation:

Low albumin: liver disease, nephrotic syndrome, malnutrition, chronic illness, or pregnancy.
High albumin: dehydration.

Potential Sources of Error:

Hemolyzed serum may give false high values.
Non-human albumin standards or contaminated reagents may cause inaccurate results.

________________________________________

References:
Ramakrishnan, Manual of Medical Laboratory Techniques, pp. 23–25.

Produce by :Dr. Yousef AL-Adbai

______________THANK_YOU_____________

Saturday, November 1, 2025

TOTAL Protein. Information about test and diagnosis of diseases

TOTAL PROTEINS.

Information about test and diagnosis of diseases

1. Purpose:

Estimation of total protein in serum/body fluids by Biuret method. Low protein levels are observed in malnutrition, acute or chronic liver diseases, nephrotic syndrome, water intoxication, salt retention syndromes, and massive intravenous infusions. Elevated protein levels are observed in dehydration due to vomiting, diarrhea, Addison’s disease and diabetic ketoacidosis. High protein levels of over 2 g/dL in body fluids are suggestive of inflammation or malignancy and are called exudates.

_______________________________________

2. Principle:

Peptide bonds of proteins in serum react with cupric ions in alkaline solutions to form a blue colored complex, the absorbance of which is measured at 578 nm. The intensity of the blue color is proportional to the amount of protein present. The reaction sequence employed in the assay of total proteins is as follows:

_______________________________________

3. Performance specifications:

3.1. Linearity: Up to 12 g/dL

3.2. Measurement range: This method has a measurement range of 5.3–8.4 g/dL of total protein in serum and body fluids.

3.3. Sensitivity: The minimum detection limit by the kit is 5.3 g/dL.

_______________________________________

4. Primary sample:

4.1. Use serum/body fluids (Pleural, Pericardial, Ascitic fluid) as specimen for the test.

4.2. Collect blood sample in a red color vacutainer tube, separate serum within 30 minutes of collection.

4.3. Process the sample on the same day within 1 hour of collection. If analysis is done on the next day, separate the serum and store it at 2–8°C for up to 30 days.

_______________________________________

5. Type of container and additive:

Use plain vacutainer tubes for collecting samples. No additive/Preservative is needed to be added.

_______________________________________

6. Reagents/Consumables:

6.1. Biuret reagent.

6.2. Total protein standard.

7. Instrument: Semi-autoanalyzer

_______________________________________

8. Procedure:

8.1 Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes.

8.2. Press “PROC”. Different test procedures will be displayed.

8.3. Select the test to be processed by entering its number and then press “ENTER” key.

8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used.

8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, if the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm.

8.6. Then feed the test samples and record the values.

8.7. Check whether the sample is hemolyzed or icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10.

_______________________________________

Assay type: End point,

Wavelength: 546 nm.

Sample volume: 10 μL,

Reagent volume: 1000 μL

Incubation time: 20 min at RT, Temperature: 37oC

_______________Dr_Yousef______________

9. Interferences:

9.1. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, If the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm.

9.2. Keep the reconstituted reagent at 2–8ºC. Discard the same if it develops precipitate.

9.3. Highly hemolytic or icteric samples, prepare sample blank by adding 1 mL of 0–9% saline to 10 microliter samples. The value of the blank is subtracted from the corresponding sample value.

_______________________________________

10. Calculating results:

11. Biological reference range:

Adults: 6.6–8.4 g/dl.

12. Critical/Alert values:

Below 5.0 g/dL and above 9.0 g/dl.

13. Laboratory interpretation:

Increase of proteins in dehydration, multiple myeloma and chronic infections (gammopathy);

decrease in malnutrition, liver diseases, nephrotic syndrome.

14. Potential sources of variability:

14.1. The reagent is linear to 12.0 g/dL. Samples with values above 10 g/dL should be diluted 1:1 with 0.9% saline, re-run, and the result multiplied by two (2)

14.2 The biuret procedure is not sensitive at low ranges (< 1 g/dL). Do not use for urine or spinal fluid.

================================

BIBLIOGRAPHY

1. Henry J, Winkelman JW. Clinical Chemistry Principles and Technique. Harper and Row, 2nd edn 1974.

2. Stricklad RD, Freeman ML, Gurule FF. Copper Binding by proteins in alkaline solution. Anal Chem 1961;33.

Reference: Manual of Medical Laboratory Techniques

Produce by: Dr. Yousef Al Adbai

Laboratorist.

______________THANK_YOU______________

triacylglycerols (triglycerides). Information about test and diagnosis of diseases

TRIACYLGLYCEROLS (TRIGLYCERIDES)

Information about test and interpretation about Result

1. Purpose:

Quantitative estimation of triacylglycerols in human serum by enzymatic method using Glycerol -3 Phosphate Oxidase (GPO) Measurement of triglycerides in conjunction with other lipid assays is used in screening the lipid status of an individual to detect atherosclerotic risks and in monitoring the response to lipid lowering measures triglyceride determinations when performed are useful in the diagnosis of primary and secondary hyperlipoproteinemia. They are also of interest in following the course of diabetes mellitus, nephrotic syndrome, biliary obstruction and various metabolic abnormalities due to endocrine disturbances.

2. Principle:

The procedure involves hydrolysis of triglycerides by lipoprotein lipase. The glycerol concentration is then determined by enzymatic assay coupled with Trinder reaction that terminates in the formation of a quinoneimine dye which is generated from 4-aminoantipyrine and 4-chlorophenol by hydrogen peroxide under the catalytic action of peroxidase. The amount of the dye formed, determined by its absorption at 500 nm, is directly proportional to the concentration of triglycerides in the sample.

3. Performance specifications:

3.1. Linearity: Up to 1000 mg/dL of serum

3.2. Measurement range: 1–1000 mg/dL of cholesterol in serum

3.3. Sensitivity: The minimum detection limit by this kit is 1 mg/dL.

4. Primary sample:

4.1. Use only fasting serum as specimen

4.2. Collect blood sample after an overnight fast of 12–14 hours when testing is a part of lipid profile

4.3. Collect 4 mL of venous blood in a plain vacutainer tube.

4.4. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes

4.5. Do not use lysed serum for testing as it may give very high results

4.6. Do not use contaminated/turbid samples for testing

4.7. Process the sample on the same day within 3 hours of collection.

4.8. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 20–25 °C for up to 2 days or at 4–8 °C for up to 7 days

5. Type of container and additive:

Use plain vacutainer tubes for collecting samples. No additive/preservative is needed to be added.

6. Reagents/Consumables:

Lipoprotein lipase, magnesium acetate, 4 aminoantipyrine, glycerol-3-phosphate oxidase, glycerol kinase, peroxidase, triglyceride standard 200 mg/dL triglycerides as triolein.

7. Instrument: Semi-autoanalyzer.

8. Procedure:

8.1. Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes.

8.2. Press “PROC”. Different test procedures will be displayed.

8.3. Select the test to be processed by entering its number and then press “ENTER” key.

8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used.

8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.300 at 520 nm if the absorbance of the ‘blank is more than 0.300, discard the reagent.

8.6. Then feed the test samples and record the values.

8.7. Check whether the sample is hemolyzed, icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or highly lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10.

_______________________________________

Assay: End point.

Reagent volume: 1000 μL

Wavelength: 546 nm

Sample volume: 10 μL

Temperature: 30°C

Zero setting with distilled water

Incubation time: 5 minutes

Conc. of standard: 200 mg/dL

_______________________________________

9. Calculating results:

10. Biological reference range:

Male: 60–165 mg/dL

Female: 40–140 mg/dL

11. Critical/Alert level values: 400 mg/dL

12. Laboratory interpretation: Triacylglycerolemia is a risk factor for myocardial infarction.

TGA is phenomenally increased in an eye disease lipemic retinalis.

13. Potential sources of variability:

13.1. Lysed serum specimens may give falsely elevated values

13.2. Do not use if the reagent is turbid as it indicates contamination of the reagent and if the absorbance of the blank reagent is more than 0.300.

================================

BIBLIOGRAPHY:

1. Annoni G, Bottasso BM. Ciaci D, Donato MF Tripoli A, Lab JJ. Res Lab Med 1982;9:115.

2. Buccolo G, David M. Clin. Chem 1973;19:476.

3. Werner M, Gabrielson DG, Estman G. Clin Chem 1981;21:268.

Reference: Manual of Medical Laboratory Techniques

Produce by: laboratorist

Dr. Yousef Al Adbai

Tuesday, October 28, 2025

LOW-DENSITY LIPOPROTEIN (LDL) CHOLESTEROL. Information about test and diagnosis of diseases

LDL (Low-Density Lipoprotein) Cholesterol — Calculation, Purpose & Reference Range

LOW-DENSITY LIPOPROTEIN (LDL) CHOLESTEROL

LDL cholesterol (LDL-C) is commonly known as the “bad cholesterol.” It plays a key role in assessing cardiovascular risk and is calculated from other lipid profile components.

1. Purpose

The estimation of LDL cholesterol in human serum is performed by calculation using a standard formula. Measuring serum LDL cholesterol helps in:

Screening lipid status to detect atherosclerotic risks
Monitoring response to lipid-lowering measures
Diagnosis and classification of hyperlipidemias

A strong relationship exists between serum LDL cholesterol and the risk of coronary heart disease.
LDL > 130 mg/dL is considered a risk factor for coronary and cerebrovascular disease and is also useful for lipoprotein phenotyping.

2. Principle

The LDL cholesterol concentration is determined by calculation rather than direct chemical assay. It uses the **Friedewald Formula**, which estimates LDL cholesterol from total cholesterol, triglycerides, and HDL levels.

3. Procedure / Calculation

The LDL cholesterol (mg/dL) is calculated as:

LDL = Total Cholesterol − (Triglycerides / 5 + HDL)

⚠️ Valid only when triglyceride level is below 400 mg/dL.

4. Reference Range

Classification	LDL Cholesterol (mg/dL)
Desirable	< 130
Borderline elevation	130 – 159
High / Elevated	> 160

Calculation valid up to triglyceride levels of 400 mg/dL.

5. Clinical Interpretation

Optimal LDL (< 100 mg/dL): Minimal atherosclerotic risk
Borderline (130–159 mg/dL): Moderate risk — lifestyle modification advised
High (> 160 mg/dL): Significant cardiovascular risk — medical intervention recommended

Critical value: LDL cholesterol above 190 mg/dL indicates severe hyperlipidemia and a high risk of coronary artery disease.

HIGH-DENSITY LIPOPROTEIN - (HDL) CHOLESTEROL. Information about test and diagnosis of diseases

HDL (High-Density Lipoprotein) Cholesterol — Test Procedure & Interpretation

HIGH-DENSITY LIPOPROTEIN — (HDL) CHOLESTEROL

Quantitative estimation of HDL cholesterol in human serum by magnesium-phosphotungstate precipitation followed by enzymatic cholesterol assay.

1. Purpose

Quantitative estimation of HDL cholesterol in human serum by precipitation method — precipitation of VLDL and LDL (using magnesium ions and phosphotungstic acid), followed by estimation of HDL cholesterol using the cholesterol esterase–cholesterol oxidase method.

Measurement of serum HDL cholesterol is useful to screen lipid status, detect atherosclerotic risk, monitor response to lipid-lowering measures, and classify hyperlipidemias. An inverse relationship exists between serum HDL cholesterol and coronary heart disease risk. HDL < 30 mg/dL is considered a risk factor for coronary and cerebrovascular disease.

2. Principle

Phosphotungstate / Mg²⁺ precipitate VLDL, LDL and chylomicrons; HDL remains in the supernatant. After centrifugation the supernatant (HDL fraction) is assayed for cholesterol using the cholesterol esterase–cholesterol oxidase enzymatic method.

Schematic:
Serum/Plasma —(Phosphotungstate / Mg²⁺)→ HDL (supernatant) + (LDL + VLDL + CM in precipitate)

3. Performance specifications

Characteristic	Value
Linearity	Up to 400 mg/dL of serum
Measurement range	1 – 400 mg/dL
Sensitivity	Minimum detection limit: 1 mg/dL
Specificity	Cholesterol oxidase oxidizes some cholesterol analogs (e.g., dihydrocholesterol, 7-dehydrocholesterol) — these analogs are not normally present in appreciable amounts in serum.

4. Primary sample

Use only fasting serum as specimen.
Collect 4 mL venous blood in a heparin vacutainer.
Allow to stand 30 minutes; separate serum by centrifugation at 2500–3000 rpm for 5–10 minutes.
Avoid icteric/hemolyzed or turbid/contaminated samples — they may give falsely high results.
Process sample the same day within 3 hours of collection. If delayed, separate serum and store at 2–8 °C for up to 7 days.

5. Container & additive

Heparin vacutainer. No preservatives required.

6. Reagents / Consumables

Precipitating reagent: phosphotungstic acid (2.4 mM) + magnesium chloride (40 mM)
Cholesterol reagent components: 4-aminophenazone, phenol
Enzymes: cholesterol esterase, cholesterol oxidase, horseradish peroxidase
Buffer (pH 6.8), stabilizers, and fillers
HDL-cholesterol standard: 50 mg/dL

7. Instrument

Semi-automatic analyzer (follow instrument specific warm-up and maintenance procedures).

8. Procedure

Bring reagents to room temperature.
Add 500 µL serum + 500 µL HDL precipitating reagent. Mix and centrifuge at 4000 rpm for 10 min to obtain a clear supernatant.
Assay the supernatant for HDL cholesterol using the cholesterol reagent (same steps as total cholesterol assay).
Instrument prep: flush tubing with 2% detergent for 2 minutes, then distilled water for 2 minutes.
Select Absorbance mode, choose the test, note reagent and sample volumes.
Run a reagent blank with each batch; blank absorbance must be < 0.100. Discard reagent if blank >= 0.100.
Feed patient samples and record values.
Check samples for hemolysis or icterus. If hemolyzed — recollect. If highly icteric or lipemic, dilute 1:10 with distilled water and multiply result by 10.

Assay details (end point):
Reagent volume: 1000 µL • Sample volume: 50 µL • Wavelength: 510 nm • Temperature: 37 °C
Zero: reagent blank • Incubation: 10 minutes • Standard concentration: 50 mg/dL

9. Interferences & precautions

Avoid blood collection in fed state — use fasting samples.
Do not use samples kept above 2–8 °C for ≥1 day.
Hemolyzed samples may give falsely elevated values; turbid reagent or high reagent blank indicates contamination — do not use.

10. Calculations

Calculate HDL cholesterol from absorbances using the standard:

Sample concentration = (Sample absorbance / Standard absorbance) × Concentration of standard

11. Biological reference range

Normal HDL cholesterol: 30 – 60 mg/dL

12. Alert / Critical values

HDL cholesterol < 30 mg/dL — considered a risk factor for coronary and cerebral vascular disease.

13. Laboratory interpretation

HDL/Total cholesterol ratio < 0.2 indicates increased coronary heart disease risk.
Equivalently, a Total cholesterol / HDL cholesterol ratio ≈ 5 is considered unfavorable.

14. Potential sources of variability

Hemolysis → falsely elevated values.
Turbid reagents or high reagent blank (≥ 0.100) → reagent contamination; do not use.

Bibliography

Castelli WP. Circulation 1977;55:767.
Castelli WP. Metabolic Therapy 1977;6:1.
Gordon T, et al. Am J Med 1977;62:707.

CHOLESTEROL TEST. Information about test and diagnosis of diseases

CHOLESTEROL TEST (CHOD-PAP METHOD)

1. Purpose

Quantitative estimation of total cholesterol in human serum by CHOD-PAP method (enzymatic photometric method). Measurement of serum cholesterol is useful in screening the lipid status of individuals to detect atherosclerotic risks and in monitoring the response to lipid-lowering measures. It is also useful in the diagnosis and classification of hyperlipidemias. Other conditions such as hepatic and thyroid diseases also influence cholesterol levels.

2. Principle

Cholesterol esters are hydrolyzed by cholesterol esterase to produce free cholesterol and fatty acids. Hydrogen peroxide is produced from the oxidation of cholesterol by cholesterol oxidase. In a coupled reaction catalyzed by peroxidase (POD), red quinoneimine dye is formed from 4-aminoantipyrine, phenol and hydrogen peroxide. The absorbance of this dye at 500 nm is proportional to the cholesterol concentration in the sample (Trinder’s reaction).

Cholesterol ester --(Chol. esterase)--> Cholesterol + Fatty acids
Cholesterol --(Chol. oxidase)--> 2 H₂O₂ + Cholesten-4-en-3-one
2 H₂O₂ + 4-Aminoantipyrine + Phenol --(POD)--> Red quinoneimine + H₂O (Red dye)

3. Performance Specifications

Linearity: Up to 1000 mg/dL of serum
Measurement range: 1 – 1000 mg/dL
Sensitivity: Minimum detection limit = 1 mg/dL
Specificity: Cholesterol oxidase is not totally specific; analogs may also be oxidized but occur rarely in serum.

4. Primary Sample

Use only plasma as specimen.
Collect 4 mL venous blood in a heparin vacutainer tube.
Centrifuge at 2500 rpm for 10 minutes.
Do not use lysed or contaminated samples.
Process within 3 hours of collection; otherwise recollect sample.

5. Container and Additive

Use plain or heparin vacutainer tubes for sample collection.

6. Instrument

Semi-auto analyzer.

7. Reagents / Consumables

4-Aminoantipyrine
Phenol
Cholesterol esterase
Cholesterol oxidase buffer (pH 6.8)
Cholesterol standard (200 mg/dL in alcohol)

8. Procedure

Switch on the analyzer and flush tubing with 2% detergent for 2 min, then with distilled water for 2 min.
Select “PROC”, choose test number, and press ENTER.
Check reagent blank (absorbance < 0.300). If higher, discard reagent.
Run test samples and record readings.
If sample is icteric or lipemic, dilute 1:10 with distilled water and multiply result × 10.

Assay Conditions:

Type: End-point
Wavelength: 510 nm
Temperature: 37 °C
Incubation time: 5 min
Reagent volume: 1000 µL
Sample volume: 10 µL
Standard concentration: 200 mg/dL

9. Interference

Avoid hemolyzed or icteric samples. Over-time reagent discoloration (light pink) is acceptable, but discard if absorbance > 0.3 OD vs distilled water at 510 nm.

10. Calculation of Results

Sample Concentration (mg/dL) = (Sample Absorbance × Standard Concentration) / Standard Absorbance

11. Biological Reference Range

Serum: 135 – 220 mg/dL
Desirable: ≤ 200 mg/dL
Borderline high: 200 – 240 mg/dL
High risk: > 240 mg/dL
Critical/Alert: > 300 mg/dL

12. Laboratory Interpretation

Hypercholesterolemia occurs in hypothyroidism (Myxedema), nephrotic syndrome, atherosclerosis, arteriosclerosis, uncontrolled diabetes, and obstructive jaundice. Hypocholesterolemia is seen in hyperthyroidism and acanthocytosis.

13. Potential Sources of Variability

Lysed plasma specimens may yield falsely elevated results.
Plasma cholesterol is stable for 7 days at room temperature and 6 months at –20 °C.
Do not use turbid reagents; this indicates contamination.

14. References

Richmond W. Clin Chem 1973; 19:1350.
Tarbutton PN, Gunter CR. Clin Chem 1974; 20:724.
Allain CC et al. Clin Chem 1974; 20:470.
Richmond W. Scand J Clin Lab Invest 1972; 29 (Suppl 26): Abst 3.25.
Young DS et al. 1975; 21D.