«Serum Matrix: Berthold Models LB 2111 and LB2104 Method: Multi-Crystal Gamma Counter with Sodium Iodide 1-1/8 x 1-1/4 Crystals Method No.: Revised: ...»
Laboratory Procedure Manual
Berthold Models LB 2111 and LB2104
Multi-Crystal Gamma Counter with
Sodium Iodide 1-1/8" x 1-1/4" Crystals
as performed by: Department of Child Health
University of Missouri-Columbia
Contact: Ms. Hsio-Mei Wiedmeyer
1-573-882-2705 Important Information for Users The University of Missouri-Columbia periodically refines these laboratory methods. It is the responsibility of the user to contact the person listed on the title page of each write-up before using the analytical method to find out whether any changes have been made and what revisions, if any, have been incorporated.
Insulin in Serum NHANES 2001–2002 Public Release Data Set Information This document details the Lab Protocol for NHANES 2001–2002 data.
A tabular list of the released analytes follows:
Lab Analyte SAS Label Number LBXIN Insulin (Uµ/mL) l10am_b LBXINSI Insulin (pmol/L) Page 2 of 17 Insulin in Serum NHANES 2001–2002
1. SUMMARY OF TEST PRINCIPLE AND CLINICAL RELEVANCE
Insulin radioimmunoassay (RIA) is a double-antibody batch method. Insulin in the specimen competes with a fixed amount of 125I-labelled insulin for the binding sites of the specific insulin antibodies. Bound and free insulin are separated by adding a second antibody, centrifuging, and decanting. The radioactivity in the pellet is then measured. The radioactivity is inversely proportional to the quantity of insulin in the specimen (1–3).
This test is used to measure insulin levels in the bloodstream and is also useful in determining pancreatic ß-cell activity.
Conditions such as obesity, a high-carbohydrate diet, and inactivity tend to increase expected normal values. Values are found to be elevated shortly after food intake and in cases of acromegaly, Cushing's syndrome, and thyrotoxicosis.
2. SAFETY PRECAUTIONSWear gloves, a laboratory coat, and safety glasses when handling all human blood specimens. Discard all plastic tips, sample cups, gloves and contaminated assay materials into a biohazard waste container.
Discard all disposable glassware into a waste container for sharps. These containers are collected and processed twice a week by the University of Missouri-Columbia (UMC) hazardous waste management personnel.
I-labelled insulin has approximately 2 µCi 125I radioactivity per kit. A laboratory coat, safety glasses, and gloves are required while handling all radioactive materials. Wear a film badge that monitors radioisotope dosage on the lapel during the RIA procedures. The work area is surveyed for contamination monthly.
Discard liquid and solid radioactive waste into their properly labeled containers. The containers are collected and disposed of by the UMC health physics/environmental health personnel.
Protect all work surfaces with disposable absorbent bench top paper, which is discarded into biohazard waste containers weekly, or whenever blood contamination occurs. Wipe all work surfaces with Envirocide solution weekly.
Material safety data sheets (MSDSs) for Envirocide are available at Diabetes Diagnostic Laboratory at the University of Missouri, Columbia (UMC).
3. COMPUTERIZATION; DATA SYSTEM MANAGEMENTThe integrity of specimen and analytical data generated by this method is maintained by the following
A. Each NHANES IV shipment is labeled with a unique container number. An electronic shipment file is sent to the laboratory at the time when samples are shipped. This file corresponds with the Shipping
Manifest Report (SMR) included in each shipment of specimens. The electronic file contains sample ID, slot ID, collection date, time, and comment code associated with each specimen. The file is formatted as a comma delimited file with an.shp extension.
B. The electronic file is saved to a network drive with a.txt extension. A backup copy is created for each file.
C. A Microsoft Access database (Hanes4.mdb) has been established on the network drive. The shipment file is first imported into a temporary import table in the database. After the data is verified with the SMR, the file is then imported into the RIA ( Insulin/C-peptide) analyte table.
D. A batch number is assigned to each shipment. A unique and sequential laboratory accession number is assigned to each specimen. A blank "Data Check Sheet" (work list) is generated by batch number and by analyte for the laboratory technologists.
E. All test result and quality control (QC) files are stored on the network server. Files are backed up daily on tape and monthly on CDs.
F. Records of specimen tracking are kept on Sample Flow Tables located in the same database.
4. SPECIMEN COLLECTION, STORAGE, AND HANDLING PROCEDURES; CRITERIA FOR SPECIMEN
REJECTIONA. Fasting blood specimens are collected for insulin analysis in accordance with NHANES IV sample collection protocol.
B. Specimen type: serum without anticoagulants or preservatives.
C. The optimal volume for this test is 1.5 mL; the minimum volume is 0.5 mL.
D. Whole blood, 3 to 5 mL, is collected in a vacuum tube (red-top or serum-separator tube). Specimens are allowed to clot at room temperature for 15–30 min, then centrifuged at 1500 × g for 10 min. Serum is transferred to a 2-mL cryogenic screw-top vial and frozen at –20°C. Frozen serum samples are sent weekly in batches in Styrofoam-insulated shipping containers with dry ice to the University of Missouri Diabetes Diagnostic Laboratory via overnight courier.
E. Insulin is stable for at least 1 month at –20°C and 1 year at –-70°C (4). Specimens should be processed within 1 hour of collection. Upon receipt, the processing laboratory will store the specimens in a –-70°C freezer until analysis. Repeated freeze/thaw cycles, except as needed for analysis, should be avoided.
F. Samples with insufficient volume or samples that arrive thawed are rejected. No analyses are performed on specimens that do not meet acceptance criteria. These conditions are noted in the assay comment codes.
5. PROCEDURES FOR MICROSCOPIC EXAMINATIONS; CRITERIA FOR REJECTION OF
INADEQUATELY PREPARED SLIDESNot applicable for this procedure.
6. EQUIPMENT AND INSTRUMENTATION, MATERIALS, REAGENT PREPARATION, CALIBRATORS(STANDARDS), AND CONTROLS A. Instrumentation (1) Berthold models LB 2111 and LB2104 multi-crystal gamma counter with sodium iodide 1-1/8" x 1crystals (Wallac Inc. Gaithersburg, MD). Counting efficiency is approximately 75% for 125I ;
the measuring range is up to 250,000 CPM per detector.
(2) Jouan refrigerated centrifuge models GR4-22 and K110 (Jouan, Winchester, VA). Temperature
range: –8°C to 60°C, temperature accuracy: 2°C, maximum RPM: 8,000 maximum, timer range:
0–99 min in 1-min increments.
(3) Eppendorf tip ejector fixed-volume pipettetors, 50 and 100-µL volume (Fisher Scientific, St. Louis.
(4) Eppendorf repeater pipette, range from 10 µL to 5 mL, precision to 0.1% (Fisher Scientific, St.
(5) Combitips for Eppendorf repeater with adapter, 2.5-mL tip graduated in 50-µL increments (Fisher Scientific, St. Louis, MO).
(6) Cornwall repeating dispenser, 2-mL volume (Fisher Scientific, St. Louis, MO).
(7) Pipetteman adjustable pipette, 200–1000 µL (Rainin Instruments, Woburn, MA).
(8) Milli-Q Plus ultrapure water system (Millipore, Bedford, MA).
B. Materials (1) Pharmacia Insulin RIA kit (Pharmacia Diagnostics AB, Uppsala, Sweden). Reagents are stable until the expiration date, which is printed on each bottle. The recommended storage temperature for all reagents is 2–8°C5. Each kit contains reagents for assaying 100 tubes. Each kit contains
Standards: 6 levels [0, 3, 10, 30, 100, and 240 micro-International Units (µIU)] of human insulin standards Diluent: 50 mL of phosphate buffer containing bovine serum albumin, EDTA, and detergent, pH 7.0.
Insulin antiserum (guinea pig): Guinea pig antiserum in assay buffer, color-coded yellow.
I-Insulin : approximately 2.6 ng (1.0 µCi at date of manufacture), color-coded blue.
Decanting suspension: Sepharose anti-guinea pig IgG raised in sheep.
Store standards at 4–8°C and mix well before use.
(2) Veronal buffer (World Health Organization (WHO), Geneva).
(3) 12- × 75-mm Borosilicate glass culture tubes (any vendor).
(4) 1.8 mL Nalgene cryogenic Vials (Nalgene Company, Rochester, NY).
(5) Racks for radioimmunoassay tubes (Fisher Scientific, St. Louis, MO).
(6) Waterproof markers for labeling tubes (any vendor).
(7) Pipette stand (Fisher Scientific, St. Louis, MO).
(8) Class A 5.0-mL volumetric pipette, calibrated "to deliver" (Fisher Scientific, St. Louis, MO).
(9) Reusable glass beakers, various volumes, accurate to 5% (Fisher Scientific, St. Louis, MO).
(10) Disposable gloves (any vendor).
(11) RIA decanting rack (Pharmacia, Uppsala, Sweden).
(12) 12-well plastic counting rack for gamma counter (Berthold, Gaithersburg, MD).
(13) Berthold multi-calibrator matched 125I sources (Berthold, Gaithersburg, MD).
(14) Prescored general purpose ampules, 1-mL (Fisher Scientific, St. Louis, MO).
(15) Aluminum foil (any vendor).
(16) Absorbent bench top paper (Whatman Lab Sales, Hillsboro, OR).
(17) Bleach (10% sodium hypochlorite) (any vendor).
(18) Viro Research Envirocide Disinfectant Decontaminant Cleaner (Fisher Scientific, St. Louis, MO).
(19) Computer printout paper (any vendor).
C. Reagent Preparation For assays of more than 100 tubes, pool kit reagents bearing identical lot numbers and mix well before use. Mix gently to avoid foaming.
D. Standards Preparation Human insulin standard materials, 0, 3, 10, 30, 100, and 240 µIU/mL, purchased from Pharmacia Diagnostics. Standards are calibrated against 1st International Reference Preparation 66/304. Ready for use.
E. Preparation of QC Materials Three levels (low, medium, high) of lyophilized human serum controls are purchased from Bio-Rad Laboratories, Irvine, CA. Using a Class A volumetric pipette, reconstitute each vial of control serum at room temperature by adding 5.0 mL distilled water. Allow the vials to stand for 10 minutes; invert the vials several times to mix contents.
Do not shake the vials. If more than one vial of each control is reconstituted, pool and mix vials with identical lot numbers together. Transfer 750-µL aliquots of each pool into cryogenic storage tubes.
Cap the tubes tightly and freeze the aliquots at –70°C. Thaw each aliquot one time only. Stable until expiration date.
The In-House control Lot # 10 (IH10) was prepared by collecting one unit of whole blood from three non-diabetic volunteers. All blood was screened for HIV and hepatitis B. Serum was separated from red blood cells, then pooled and transferred 750 µL aliquot into cryogenic storage tubes. The tubes were tightly capped and frozen at –70°C. Reconstitution of IH10 is not required. Thaw each aliquot one time only.
7. CALIBRATION AND CALIBRATION VERIFICATION PROCEDURES
(3) The calibration curve is displayed immediately following the standard curve summarization. To verify the mathematical fit, the smoothing factor must be less than 16 for assay acceptance.
(4) Percent B0/total counts (TC) is monitored to verify the binding activity of the antibody and labeled I insulin solution. It should be between 45% to 55%. If the B0/TC value is outside of those limits, notify the supervisor prior to accepting a run.
B. Verification The World Health Organization's (WHO's) international standard for insulin (first International Reference Preparation, 66/304) is used monthly to verify the insulin calibration curve. Prepare the stock solution by dissolving the ampoule supplied by WHO in 0.75 mL of 0.07 M veronal buffer (pH 8.6). Class A volumetric pipettes are used to prepare subsequent working standards of 3, 9, 30, 100, 120, 150 and 240 µIU/mL with Pharmacia diluent (WHO Lot#2, prepared at the University of Missouri in November 1997). Store both stock and working standards in sealed 1-mL ampule vials at –20°C.
The stock and working solutions are stable at –20°C for years. Thaw one set of working standard monthly and, in a regular insulin assay, analyzes it simultaneously with Pharmacia standard. The
validation is monitored in two areas:
(1) The Pharmacia kit standard curve is compared directly with WHO Lot #2 insulin standard curve.
The relationship between the two standard curves over time should be consistent with the initial relationship established in November 1997.
(2) A group of samples is analyzed by using both sources of standards. The means and linear regression relationships between the paired insulin values are calculated and charted. For low levels of insulin (20 µIU/mL), the mean insulin value obtained from Pharmacia should be within 25% of the mean value obtained from WHO. For medium (20 to 60 µIU/mL) and high (60 µIU/mL) levels of insulin, the mean insulin values from Pharmacia should be within 15% of the mean values from WHO.
8. PROCEDURE OPERATING INSTRUCTIONS; CALCULATIONS; INTERPRETATION OF RESULTS
A. Preliminaries (1) Allow frozen samples and quality control materials (three levels of Bio-Rad controls and one level of in-house control) to reach ambient temperature. Invert each gently to mix.