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1) PRODUCT {Test} NAME 2) INTENDED USE and TEST TYPE (qualitative or qualitative) 3) SUMMARY AND EXPLANATION 4) PRINCIPLES OF THE PROCEDURE {NCCLS lists SAMPLE COLLECTION/HANDLING next} 5) REAGENTS (name/concentration; warnings/precautions; preparation; storage; environment; Purification/treatment; indications of instability) 6) INSTRUMENTS required – Refer to Operator Manual (... for equipment for; use or function; Installation; Principles of operation; performance; Operating Instructions; Calibration* {*is next in order for NCCLS – also listed in “PROCEDURE”}’ precautions/limitations/hazards; Service and maintenance information 7) SAMPLE COLLECTION/HANDLING 8) PROCEDURE {NCCLS lists QUALITY CONTROL (QC) next} 9) RESULTS (calculations, as applicable; etc.) 10) LIMITATIONS/NOTES/INTERFERENCES 11) EXPECTED VALUES 12) PERFORMANCE CHARACTERISTCS 13) BIBLIOGRAPHY (of pertinent references) 14) NAME AND PLACE OF BUSINESS OF MANUFACTURER 15) DATE OF ISSUANCE OF LABELING (instructions)

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Form 364 Helena Laboratories 1/2006 (Rev 3) 2. Acid Violet Stain Ingredients: The stain contains Acid Violet. WARNING: FOR IN-VITRO DIAGNOSTIC Cat. No. 3430 USE ONLY. DO NOT INGEST. Preparation for Use: Dissolve the dry stain in 1 L 10% acetic acid. Filter before use if necessary, then fill the stain vat. The SPIFE 3000 SPE Hi Res-20 method is Storage and Stability: The dry stain should be intended for the qualitative separation of stored at 15 to 30°C and is stable until the protein fractions in serum, cerebrospinal fluid expiration date indicated on the package. The (CSF) or urine using agarose gel stain solution is stable for six months when electrophoresis on the SPIFE 3000 system. stored at 15 to 30°C in a closed container. SUMMARY Signs of Deterioration: The diluted stain High resolution electrophoresis achieves should be an homogeneous mixture free of better resolution of the proteins beyond the precipitate. classical five band patterns, thereby increasing 3. Citric Acid Destain the diagnostic usefulness of protein patterns.1-3 Ingredients: After dissolution, the destain Approximately fifteen serum proteins have contains 0.3% (w/v) citric acid. been studied extensively because they may be WARNING: FOR IN-VITRO DIAGNOSTIC measured easily.4-7 In this context, high USE. DO NOT INGEST – IRRITANT. resolution electrophoresis refers to systems which separate 95% of the total protein mass into 10 to 15 discrete fractions. PRINCIPLE Proteins are large molecules composed of covalently linked amino acids. Proteins can be either polar or nonpolar at a given pH depending on electron distributions resulting from covalent or ionic bonding of struc- tural subgroups. In the Helena procedure, proteins are separated accord- ing to their respective electrical charges on agarose gel, using both the electrophoretic and electroendosmotic forces present in the system. The separations are stained with a protein sensitive stain. COMPONENTS 1. SPIFE SPE Hi Res Gel Ingredients: Each gel contains agarose in Tris barbital buffer, sorbitol and a preservative. WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO NOT INGEST. The gel contains barbital which, in sufficient quantity, can be toxic. Preparation for Use: The gels are ready for use as packaged. Storage and Stability: The gels should be stored at room temperature (15 to 30°C) and are stable until the expiration date indicated on the package. The gels must be stored in the protective packaging in which they are shipped. DO NOT REFRIGERATE OR FREEZE THE GELS. Signs of Deterioration: Any of the following conditions may indicate deterioration of the gel: (1) crystalline appearance indicating the agarose has been frozen, (2) cracking and peeling indicating drying of the agarose, (3) bacterial growth indicating contamination, (4) thinning of the gel blocks. ® Urine: Urine samples may be run neat, SPIFE 3000 SPE Hi diluted or concentrated. One application or Res-20 three applications can be made to obtain desired intensity. To concentrate urine, shake Procedure for Plastic samples to homogenize. Centrifuge desired volume at 2000 x g for 5 minutes. Remove Applicators supernatant and concentrate as follows: Total Protein (mg/dL) Conc. Factor < 50 100x 50-100 50x Preparation for Use: Pour 11 L of 100-300 25x deionized water into the Destain vat. 300-600 10x Add the entire package of Destain. > 600 5x Mix well until completely dis- solved. CSF: CSF samples may be used after proper Storage and Stability: Store the concentration of 10-50x. Destain at 15 to 30°C. It is stable until PROCEDURE the expiration date on the package. Materials provided: The following materials Signs of Deterioration: Discard if needed for the procedure are contained in the solution becomes cloudy. SPIFE SPE Hi Res-20 Kit (Cat. No. 3430). INSTRUMENT Individual items are not available. A SPIFE 3000 Analyzer must be used to SPIFE SPE Hi Res-20 Gels (10) apply sample, electrophorese, stain, Acid Violet destain and dry the gel. Refer to the Stain (1 appropriate Operator’s Manual for vial) REP detailed instructions. Blotter C SPECIMEN COLLECTION AND (10) HANDLING Citric Acid Destain (1 Specimen: The specimen may be pkg) Blade serum, cerebrospinal fluid (CSF) or Applicator urine. Serum specimens should be free Kit (10) of hemolysis or lipemia. A fibrinogen Materials provided by Helena band, which may obscure the beta- Laboratories but not contained in the kit: gamma zone, will appear in plasma Item Cat. No. samples. Storage: Fresh serum is the specimen SPIFE 3000 Analyzer 1088 Hi Res Protein Marker 5141 of choice. If storage is necessary, Gel Block Remover 1115 samples may be stored covered at 2 to 8°C for 48 hours. REP Prep 3100 Specimen Preparation: Applicator Blade Weights 3387 Serum: Dilute serum samples 1:2 with Disposable Sample Cups 3369 0.85% saline solution (1 part serum and SPIFE Dispo Cup Tray 3370 1 part 0.85% saline solution). Materials needed but not provided: 10% Acetic Acid 0.85% Saline Solution STEP-BY-STEP IV. Sample Application/Electrophoresis METHOD N Se Uri Hi E o. Re I.Sample of ru ne l Preparation m s A Ma e p CSF rke c pl 1:2 r ic t wit neat,dil at nea r io h uted t n sal or o s ine concent p 1 rated h o r e s i s

U n i t • Serum and/or Urine 1) No Prompt Load Sample 1 00:30 21°C SPD1 3 nea 1:3 2)No Prompt 1. S t,di wit Apply Sample 1 e lut r h u ed m sali 00:30 10- ne 50x 21°C or con SPD1 cen trat LOC1 ed Dilute each serum samples. If one sample 1:2 with application of 0.85% saline urine is used, the solution (1 part marker should be serum and 1 part run neat. 0.85% saline However, if three solution). The Hi applications of Res Marker should urine are used, be run neat. Only the marker should one application is be diluted 1:3 used. with saline. Three 2.Urine applications of Urine samples urine cannot be may be run neat, run with the diluted or serum specimens. concentrated as 3.CSF described in CSF samples "Specimen should be Preparation". concentrated 10- One application of 50x. The marker urine can be run should be diluted on the same gel 1:3 with saline. with serum Three applications o e de into the vertical 3) No Prompt f D slot numbered 8 in Electrophoresis 1 C i the Applicator S s Assembly. 18:00 F p NOTE: The 17°C a o Applicator Blade s r will only fit into 525 V e a the slots in the n b Applicator 90 mA e l Assembly one 4) Remove gel e e way; do not try blocks, (continue) d A to force the Dry 1 p Applicator Blade e into the slots. d p 12:00 a li n c 54°C 5)N d a t o c o a r P n B r - l o n a m o d p t t e

b f e E r N r o u D m n O w t F it h h e T s p E e a S r c T u k • Urine/CSF (3 m a applications) g 1) No Prompt Load Sample 1 s i p n 00:30 e g c . 21°C i 2. P m l SPD1 e a 2) No Prompt n c Apply Sample 1 s e . t 00:30 II. h Instr e 21°C um A SPD1 ent p Set p LOC1 up li 3) No Prompt 1. R c Load Sample 2 e a m t 00:30 o o v r 21°C e B o l SPD1 n a 4) No Prompt A C 1:00 p 17°C p S 21°C l P 8) No Prompt 525 V y D Electrophoresis 1 1 90 mA S 6) N 18:00 9) Remove gel a o blocks, (continue) m p P 3. Place an 10)Dry 1 12:00 l r Applicator Blade 54°C e o Weight on top of N m the Applicator o 2 p Blade. When t placing the weight P r 0 A on the blade, o 0 p position the weight : p m with the thick side p 3 l to the right. 0 y t

2 S E 1 a N ° m D C p l O S e F P D 3 T 1 E 1 S L : T Staine O 0 r Unit C 0 4. Slide the lay the gel down 1 Disposable Sample on the REP Prep, 5) N 2 Cups into the starting from the o 1 middle row, left side and ° P C numbered 21 to ending on the r 40, of the Cup right, fitting the o S Tray. obround hole m P 5. Pipette 20 µL of over the right pin. p D the appropriate Use lint-free t 1 sample into the tissue to wipe L cups. Cover the around the edges o L tray until ready for of the plastic gel a O use. backing, d C III. Gel Preparation especially next to 1 1. Remove the gel electrode posts, S 7) N from the to remove excess a o m protective REP Prep. Make p P packaging and sure no bubbles l r discard overlay. remain under the e o 2. Dispense gel. m approximately 2 4. Place a SPIFE 3 p mL of REP Prep Blotter C on the t onto the left side gel with the 0 A of the longer edge 0 b electrophoresis parallel with gel : s chamber. blocks. Gently 3 o 3. Place the left edge blot the entire 0 r of the gel over the b surface of the gel 2 REP Prep aligning using slight 1 1 the round hole on fingertip pressure ° the left pin of the on the blotter, and chamber. Gently remove the b s e ledge of each gel • Serum, CSF l e block outside the and Urine o . magnetic posts. 1) No Prompt t W Improper contact Stain 1 t i between electrode e p and the gel block 4:00 r e may cause skewed . w patterns. Close the REC = OFF 5. C i chamber lid. VALVE = 5 l t 7.Press the TEST 2) No Prompt e h SELECT/CONTI Destain 1 a NUE button n a located on the 3:00 t l Electrophoresis h i and Stainer sides REC = ON e n of the instrument e t until the HIGH VALVE = 2 l f RESOLUTION 3) No Prompt e r option appears on Destain 2 c e the display. t e 2:00 r t REC = ON o i d s VALVE = 2 e s 4) No Prompt s u Destain 3 w e it . 1:00 h 6. P d l REC = ON e a i c VALVE = 2 5) No Prompt o e Dry 1 n a i c 12:00 z a e r 63°C d b 6)N w o o a n t P e e r r l o b e m p e c t f t o r E r o N e d D a e n o O d n F a f t T t h E e e S T r o 1. Open the e u chamber lid and a t place the Cup c s Tray with h i samples on the u d SPIFE 3000. A C ress the START/ and use the Gel l lo STOP button. An Block Remover i s option to either to remove the g e begin the test or gel blocks. Place n th skip the opera- one electrode e tion will be across each end t c presented. Press of the gel to h h START/STOP prevent curling e a button to begin. during drying. m The SPIFE 3000 Dispose of h b will apply the blades and cups o er samples, as biohazardous l li electrophorese waste. e d. and beep when 4. Close the s 2. W completed. chamber lid, and i 3. After press the TEST i t electrophoresis is SELECT/CONTI n h complete, open NUE the chamber lid button to dry the t t gel. h h e e t a r p a p y r o w p i r t i h a t t e h e t e p s i t n s n a o m n e t o h n e t i h n e s t d r i u s - p l m a e y n , t . p V. Visualization α from 55 to 75% of the normal CSF protein. The 1 band 1. After the gel has been dried, open the chamber lid α α consists primarily of 1-antitrypsin, the -lipoprotein and carefully remove the gel from the α fraction being greatly decreased. The 2 region is not a electrophoresis chamber. dominant fraction, as with plasma, owing to relative 2. Remove the Gel Holder from the stainer unit. α decreases in large proteins such as 2-macroglobulin Attach the gel to the holder by placing the round and the polymeric haptoglobin phenotypes. Transferrin hole in the gel backing over the left pin on the β β is detected in the 1 region and the major 2 protein is a holder and the obround hole over the right pin on carbohydrate-deficient "CSF-specific" transferrin. The the holder. gamma region, consisting almost exclusively of 3. Place the Gel Holder with the attached gel facing immunoglobulin G can show some very faint banding in backwards into the stainer unit. normal samples. The cathodal end of this zone often 4. With the appropriate test name on the display, contains a low-Mr non-immunoglobulin protein, referred press the START/ STOP button. An option to either to as gamma trace, which is perhaps synthesized within begin the test or skip the operation will be the central nervous system, but has undetermined presented. Press START/STOP to begin. The clinical significance. instrument will stain, destain and dry the gel. LIMITATIONS 5. When the gel has completed the process, the Aging of serum samples will cause the C3 band to instrument will beep. Remove the Gel Holder from migrate in the transferrin region. Fresh specimens the stainer. only should be tested, and they should not be hemolyzed Stability of End Product: or lipemic. Samples should be at room temperature The completed SPIFE 3000 SPE Hi Res-20 Gel is stable before use to prevent cryoprecipitation at the for an indefinite period of time. application point. Gels which do not lay flat in the Quality Control: chamber, or those with surface artifacts, should not be The Hi Res Protein Marker (Cat. No. 5141) may be used used. to verify appropriate protein band separation and stain INTERPERTATION OF RESULTS sensitivity. Refer to the package insert for more Serum information. High resolution protein electrophoresis patterns are RESULTS primarily interpreted by comparing the relative Plasma Proteins intensities of the bands obtained on unknown Figure 1 shows the relative position of most of the specimens with those obtained on known normal plasma proteins. individuals. One of the most common abnormal serum Prealbumin protein patterns is that observed in the non-specific inflammatory response which is characterized by an Albumin α increase in 1-antitrypsin and haptoglobin with decreased prealbumin, albumin and transferrin. While it is not useful in establishing a general ( (If o Ceruloplasmi present r -split produc o n t) s o Hemopexi I m g u n A c C 3 o I i c (I g d f M ) p re I α s g e G 1 nt - A s Figure 1: Illustration c pl of relative band i it d p positions ro Urine Proteins G d u l ct y )

C 3 d α Lipo di important variations microglobulin, a strong proteinuria, tubular- α Antitrypsin 1 ag are: band in the mid-beta type proteinuria as well Haptoglobin no - elevation of the β as mixed glomerular- region due to 2- (Phenotype si transferrin band, microglobulin and tubular patterns and Dependent) s, suggesting a low sometimes diffuse the various overflow α 2 it level of iron background staining in states can be easily Macroglobulin - presence of Transferrin is the gamma region due distinguished and C3 (Native) us monoclonal to free light chains. characterized, thus β-Lipoprotein ef proteins, Chronic renal disease providing useful Fibrinogen (Plasma) ul suggesting or renal failure can information on specific Application Point in abnormalities of lead to damage of both functions within the m the immune glomerulus and tubules. nephron. on system This results in a CSF it - low haptoglobin, combined pattern with One of the primary or suggesting both “glomerular-type” reasons for performing in elevated RBC and “tubular-type” high resolution protein g turnover or in- proteins appearing in electrophoresis is for a vitro hemolysis the urine. the detection of pa - CRP presence, Cerebrospinal Fluid oligoclonal bands in ti indicating an Proteins cerebrospinal fluid.9,10 en acute When CSF is Oligoclonal t’s inflammatory concentrated, the immunoglobulin re response pattern from a normal patterns can be seen in sp - low prealbumin, adult shows a both serum and on albumin and prominent pre-albumin cerebrospinal fluid as se transferrin with fraction that migrates faint bands in the to diffuse slightly faster than gamma zone. They are th hypergam- plasma prealbumin. usually multiple er maglobulinemia, Albumin is the major homogeneous, narrow ap suggesting band on and discrete in y. chronic electrophoresis, appearance. When seen Ot inflammation, comprising in serum, the bands he infection or anti- indicate the presence r genic stimulation of immune complexes

ex - low C3 on fresh which are associated a samples, with Hodgkins disease m suggesting or a non-specific early pl complement immune response to es consumption. various disease states. of Urine In the cerebrospinal cli High resolution fluid, their presence is ni protein important supportive ca electrophoresis is an evidence for the lly excellent analytical diagnosis of multiple tech- nique to gain a scle- rosis (MS) in the broad overview of proper clinical setting. urine proteins.3,8 The oligoclonal bands Glomerular-type in serum are not An electrophoretic β indicative of MS. The in the 1 region. The pattern of normal urine serum pattern shows presence of the will show a trace of marked decreases in oligoclonal pattern is a albumin sensitive test for MS and sometimes a faint these proteins with increases in the large because it is present in transferrin band. more than 90% of MS The urine pattern in proteins which are retained by the patients at some time glomerular proteinuria during the course of usually consists of glomerulus. The urine pattern in the disease. It may be strong bands of less sensitive α tubular proteinuria albumin, both 1 acid α usually consists of a glycoprotein and 1- antitrypsin in a broad faint albumin band, a double band in the α α zone and transferrin 2 1 α region due to 2- during an initial attack and is not completely specific for MS. However, it is usually not difficult to distinguish between MS and the other diseases most commonly associated with an oligoclonal pattern, such as infec- tions of the central nervous system, syphilis, cryptococcus, toxoplasmosis, subacute sclerosing panencephalitis and Guillian-Barre syndrome.9

BIBLIOGRAPHY 1. Laurell, C.B., Compostion and Variation of the Gel Electrophoreic Fractions of Plasma, Cerebrospinal Fluid and Urine. Scand J Clin Lab Invest 29 Suppl 24:71, 1972. 2. Killingsworth, L.M. et al., Protein Analysis, Deciphering Cerbrospinal Fluid Patterns, Diag Med 1-7, March/April, 1980. 3. Killingsworth, L.M. et al., Protein Analysis, Finding Clues to Disease in Urine, Diag Med 69-75, May/ June, 1980. 4. Killingsworth, L.M., Clinical Applications of Protein Determinations in Biological Fluids Other Than Blood, Clin Chem 28(5):1093-1102, 1982. 5. Ritzmann, S.E. and Daniels, J.C., Diagnostic Proteinology: Separation and Characterization of Proteins, Qualitative and Quantitative Assays, in Laboratory Medicine, Harper and Row, Inc., Hagerstown, 1979. 6. Killingsworth, L.M. et al., Protein Analysis, Diag Med, 3-15, Jan/Feb., 1980. 7. Killingsworth, L.M., Plasma Protein Patterns in Health and Disease, CRC Critical Reviews in Clinical Laboratory Sciences, August, 1979. 8. Peterson, P.A., Ervin, P.E., and Beggard, I., Differentiation of Glomerular, Tubular and Normal Proteinuria: Determinations of Urinary Excretion of α2- microglobulin, Albumin and Total Protein, J Clin Invest, 48:1189-1198, 1969. 9. Gerson, B., Two Agarose Electrophoretic Systems for Demonstration of Oligoclonal Bands in Cerebrospinal Fluid Compared, Clin Chem 26(2): 343- 345, 1980. 10.Brett, E., Oligoclonal Banding Detects MS, Clin Chem News, 10-11, Nov., Pro 259 2/16 1980. Beaumont, Texas USA 77704

For Sales, Technical and Order Information, and Service Assistance, call 800-231-5663 toll free. Helena Laboratories warrants its products to meet our published specifications and to be free from defects in materials and workmanship. Helena’s liability under this contract or oth- erwise shall be limited to replacement or refund of any amount not to exceed the purchase price attributable to the goods as to which such claim is made. These alternatives shall be buyer’s exclusive remedies. In no case will Helena Laboratories be liable for consequential damages even if Helena has been advised as to the possibility of such damages. The foregoing warranties are in lieu of all warranties expressed or implied including, but not limited to the implied warranties of merchantability and fitness for a particular purpose.