BIBLIOGRAPHY ® 1. Wintrobe, Maxwell M., Clinical Hematology, 6th Edition, SPIFE 4000 Acid LeFebiger, Philadelphia, 1967. 2. Fairbanks, V.F., Diagnostic Medicine, Nov/Dec., 53-58, Cat. No. 2322 Procedure 1980. 3. Schneider, R.G., et al., Laboratory Identification of the , Lab Management, August, 29-43, 1981. The SPIFE 4000 Acid Hemoglobin method is intended for the Storage and Stability: The gels should be stored horizontal- 4. Center for Disease Control, Laboratory Methods for qualitative determination of hemoglobins using agar in acidic ly at room temperature (15 to 30°C) and are stable until the Detecting Hemoglobinopathies, U.S. Department of Health buffer on the SPIFE 4000 system. expiration date indicated on the package. The gels must be and Services/Public Health Service, 1984. SUMMARY stored in the protective packaging in which they are shipped. 5. Schneider, R.G., Methods for Detection of Hemoglobin Hemoglobins (Hb) are a group of whose chief func- DO NOT REFRIGERATE OR FREEZE THE GELS. Variants and Hemoglobinopathies in the Routine Clinical tions are to transport oxygen from the lungs to the tissues and Signs of Deterioration: Any of the following conditions may Laboratory, CRC Critical Reviews in Clinical Laboratory carbon dioxide in the reverse direction. They are composed of indicate deterioration of the gel: (1) crystalline appearance Sciences, 1978. polypeptide chains, called , and iron protoporphyrin indicating the agarose has been frozen, (2) cracking and 6. Schneider, R.G., et al., Abnormal Hemoglobins in a Quarter groups. A specific sequence of amino acids constitutes each peeling indicating drying of the agarose, (3) bacterial growth Million People, Blood, 48(5):629-637, 1976. of four polypeptide chains. Each normal hemoglobin molecule indicating contamination, (4) thinning of the gel blocks. 7. Huisman, T.H.J. and Schroeder, W.A., New Aspects of the contains one pair of alpha and one pair of non-alpha chains. 2. Acid Blue Stain Structure, Function, and Synthesis of Hemoglobins, CRC The non-alpha chains of are called gamma. Ingredients: When dissolved as directed, the stain contains Press, Cleveland, 1971. 0.5% (w/v) acid blue stain. A minor (3%) hemoglobin fraction called HbA2 contains alpha 8. Schmidt, R.M., et al, The Detection of Hemoglobinopathies, and delta chains. Two other chains are formed in the embryo. WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO CRC Press, Cleveland 1974. The major hemoglobin in the erythrocytes of the normal adult NOT INGEST. 9. Weatherall, D.J. and Clegg, J.B., The Thalassaemia Preparation for Use: Dissolve the dry stain (entire contents is HbA, but there are small amounts of HbA2 and HbF. In Syndromes, Blackwell Scientific Publications, Oxford, 1972. addition, over 400 mutant hemoglobins are now known, some of vial) in 1 L of 5% glacial acetic acid. Mix thoroughly for 30 10. Lehman, H. and Huntsman, R.G., Man’s Haemoglobins, of which may cause serious clinical effects, especially in the minutes. J.B. Lippincott Co., Philadelphia, 1974. homozygous state or in combination with another abnormal Storage and Stability: The dry stain should be stored at 15 hemoglobin. Wintrobe1 divides the abnormalities of hemoglobin to 30°C and is stable until the expiration date indicated on the For Sales, Technical and Order Information and synthesis into three groups: package. The diluted stain is stable one year when stored at Service Assistance, call 800-231-5663 toll free. (1) Production of an abnormal molecule (e.g. sickle cell 15 to 30°C. anemia) Signs of Deterioration: The diluted stain should be a homo- Helena Laboratories warrants its products to meet our published specifications (2) Reduction in the amount of normal protein synthesis (e.g. geneous mixture free of precipitate. Discard if precipitate and to be free from defects in materials and workmanship. Helena’s liability under forms. The stain must be replaced after processing ten gels this contract or otherwise shall be limited to replacement or refund of any amount to avoid contamination. not to exceed the purchase price attributable to the goods as to which such claim (3) Developmental anomalies (e.g. hereditary persistence of is made. These alternatives shall be buyer’s exclusive remedies. fetal hemoglobin (HPFH) 3. Hemolysate Reagent In no case will Helena Laboratories be liable for consequential damages even if The two mutant hemoglobins most commonly seen in the Ingredients: The reagent contains deionized water with 0.005 Helena has been advised as to the possibility of such damages. United States are HbS and HbC. Hb Lepore, HbE, HbG- M EDTA, 0.175% saponin and 0.07% potassium cyanide. The foregoing warranties are in lieu of all warranties expressed or implied includ- ing, but not limited to, the implied warranties of merchantability and fitness for a Philadelphia, HbD-Los Angeles and HbO-Arab may be seen WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO particular purpose. less frequently.2 NOT PIPETTE BY MOUTH. The reagent contains potassium Electrophoresis is generally considered the best method for cyanide. separating and identifying hemoglobinopathies. The protocol Preparation for Use: The reagent is ready for use as pack- for hemoglobin electrophoresis involves stepwise use of two aged. systems.3-8 Storage and Stability: The reagent should be stored at room Initial electrophoresis is performed in alkaline buffers. Cellulose temperature (15 to 30°C) and is stable until the expiration acetate was the major support medium used because it date indicated on the vial. yields rapid separation of HbA, F, S and C and many other Signs of Deterioration: Discard if solution has precipates or mutants with minimal preparation time. However, because flocculent. of the electrophoretic similarity of many structurally different 4. Citric Acid Destain hemoglobins, the evaluation must be supplemented by citrate Ingredients: After dissolution, the destain contains 0.3% agar electrophoresis which measures a property other than (w/v) citric acid. electrical charge. WARNING: FOR IN-VITRO DIAGNOSTIC USE. DO NOT This method is based on the complex interactions of the INGEST - IRRITANT. hemoglobin with an acid electrophoretic buffer and the agar Preparation for Use: Pour 11 L of deionized water into the support. The SPIFE 4000 Acid Hemoglobin method is a simple Destain vat. Add the entire package of Destain. Mix well until procedure requiring minute quantities of hemolysate to provide completely dissolved. Pour the entire contents of the Destain a screening method for the presence of abnormal hemoglobins Additive bottle into the prepared Destain and mix. such as HbS, HbC and HbF. Storage and Stability: Store the Destain at 15 to 30°C. It is PRINCIPLE stable until the expiration date on the package. Very small samples of hemolysates prepared from washed, Signs of Deterioration: Discard if solution becomes cloudy. packed cells are automatically applied to the SPIFE 4000 5. Destain Additive Acid Hb gel. The hemoglobins in the sample are separated by Ingredients: The product is a wetting agent. electrophoresis using a citrate buffer and are stained with Acid WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO Blue Stain. NOT INGEST - IRRITANT. REAGENTS Preparation for Use: Pour the entire contents of the Destain 1. SPIFE 4000 Acid Hb Gel Additive bottle into 11 L of prepared Citric Acid Destain. Ingredients: Each gel contains agar in citrate buffer with Storage and Stability: The additive should be stored at 15 0.25% EDTA and thimerosal as a preservative. to 30°C and is stable until the expiration date indicated on the package. Beaumont, Texas USA 77704 Pro. 212 Preparation for Use: The gels are ready for use as packaged. 4/19(4) Shaded areas indicate that the text has been modified, added or deleted. Signs of Deterioration: The additive should be free of precipitate. 7. Using the prompts, prime the surfactant delivery system IV. Electrophoresis REFERENCE VALUES INSTRUMENT according to the instructions in the Operator’s Manual. 1. Click START on the screen and respond to the analyzer At birth, the majority of hemoglobin in the erythrocytes of the A SPIFE 4000 must be used to apply samples, electrophorese, II. Sample and Gel Preparation prompts. The analyzer will apply samples, electrophorese, normal individual is fetal hemoglobin, HbF. Some of the major stain, destain and dry the gel(s). stain, destain and dry the gels. Refer to the Operator’s Manual 1. Prepare hemolysates of patient specimens and controls as adult hemoglobin, HbA, and a small amount of HbA2 are also for detailed instructions. in­structed in the “Specimen Preparation”­ section. 2. The cassette with the gel will be dropped into the cas- present. At the end of the first year of life and through adult- SPECIMEN COLLECTION AND HANDLING 2. With the notch in the SPIFE 4000 White sample tray on the sette receptacle. hood, the major hemoglobin present is HbA with up to 3.5% 3. Remove the cassette(s) from the receptacle. If gel stor- Specimen: Whole blood collected in EDTA tubes is the spec- left, hand pipette 40 µL of patient or control hemolysate HbA2 and less than 2% HbF. imen of choice. into each sample cup in the top row. Load samples in age is required, remove and discard the two gel blocks. INTERPRETATION OF RESULTS Specimen Storage: If storage is necessary, whole blood and cups 1-7, skip the smaller cup in the center of the row Clean or wipe the non-gel side. If not, discard the used Most cause no discernible clinical symp- packed cells may be stored up to 1 week at 2 to 8°C. Frozen and then load samples in cups 8-14. gels, applicator blades and sample trays as biohazard- toms, so are of interest primarily to research scientists. Variants samples may produce an artifact band between HbF and HbA, 3. Stack the appropriate number of disposable sample trays, ous waste. are clinically important when their presence leads to sickling and band intensity may diminish, especially with . with samples loaded, into the sample tray holder (one 4. Cassettes and carbon electrodes should be washed and disorders, thalassemia syndromes, life long cyanosis, hemolytic Specimen Preparation: Washed, packed cell hemolysates tray per gel), placing the first tray on the bottom. Place an dried after each use with deionized water. Refer to the anemias or erythrocytosis or if the heterozygote is of sufficient must be prepared for each patient specimen. additional empty sample tray on top of the stack to prevent Operator’s Manual for instructions. prevalence to warrant genetic counseling. The combinations A) Whole Blood sample evaporation of sample. Evaluation of the Hemoglobin Bands of HbSS, HbSD-Los Angeles and HbSO-Arab lead to serious 1. Centrifuge anticoagulated blood for 10 minutes to sepa- 4. Carefully open one end of the pouch, remove the gel from The hemoglobin gel should be inspected visually for the pres- sickling disorders.2 Several variants including HbH, E-Fort rate cells from plasma. the protective packaging and discard the overlay. ence of abnormal hemoglobin bands. Worth and Lepore cause a thalassemic blood picture.2 The two 2. Remove plasma. 5. Using a SPIFE 4000 Blotter C, gently blot the entire gel. migrates with HbF. The Helena AFSC Hemo Control provides variant hemoglobins of greatest importance in the U.S., in terms 3. Wash packed cells 3 times by resuspending in 5 to 10 Discard the blotter. a marker for band identification. of frequency and pathology, are HbS and HbC.2 Sickle cell volumes of normal saline solution (0.85% NaCl), centri- 6. Hold the gel so that the barcode is at the top. Place the gel Stability of End Product: The dried gels are stable for an anemia (HbSS) is a cruel and lethal disease. It first manifests fuging and aspirating supernatant. into the cassette by holding the gel backing in one hand indefinite period of time. itself at about 5 to 6 months of age. The clinical course presents 4. After washing the samples, prepare the samples by mix- and gently bending the gel. Slide each end of the gel back- Quality Control: The Helena AFSC Hemo Control (Cat. No. agonizing episodes of pain and temperature elevations with ing 10 µL sample to 100 µL Hemolysate Reagent. Vortex ing into the slots of the cassette to hold it in place. Align 5331) should be run on each SPIFE 4000 Acid Hemoglobin anemia, listlessness, lethargy and infarct in virtually all organs or shake vigorously for 15 seconds. the cutout in the gel backing with the alignment pin in the Gel. The control verifies all phases of the procedure and acts of the body. The individual with homozygous HbCC suffers B) Control cassette. as a marker to aid in the identification of the hemoglobins in mild hemolytic anemia which is attributed to the precipitation or AFSC (Cat. No. 5331) 1:2 (1 part control + 1 part Hemolysate 7. Ensure the gel blocks make good contact with the elec- the unknown samples. crystallization of HbC within the erythrocytes. Cases of HbSC Reagent) trodes to prevent skewed patterns. RESULTS disease are characterized by hemolytic anemia that is milder Figure 1 illustrates the electrophoretic mobility of bands on the PROCEDURE 8. Place the cassette with the gel into the humidor and cover than sickle-cell anemia. SPIFE 4000 Acid Hemoglobin Gel. Materials provided: The following materials needed for the the topmost cassette with the Gel Staging Lid. Close the The are a group of hemoglobin disorders charac- procedure are contained in the SPIFE 4000 Acid Hemoglobin humidor lid to minimize gel dehydration. terized by hypochromia and microcytosis due to the diminished Kit (Cat. No. 2322). Individual items are not available. 9. Repeat Steps 4-8 for each gel needed. synthesis of one globin chain (the α or β) while synthesis of the 9,10 SPIFE 4000 Acid Hemoglobin Gels (10) NOTE: A maximum of 3 gels can be run at a time. other chain proceeds normally. This unbalanced synthesis Acid Blue Stain (1 vial) III. Electrophoresis Parameters results in unstable globin chains. These precipitate within the Hemolysate Reagent (25 mL) Using the instructions provided in the Operator's Manual, red cell, forming inclusion bodies that shorten the life span Citric Acid Destain (1 pkg) select of the cell. In α-thalassemias the α-chains are diminished or SPIFE 4000 Blotter C (10) -- Test Type: Acid Hb absent, and in the β-thalassemia the β-chains are affected. SPIFE 4000 Applicator Blades (10) Test Name: Acid Hb Another quantitative disorder of hemoglobin synthesis, hered- Destain Additive (28 mL) Check the programmed parameters for each of the follow- itary persistent fetal hemoglobin (HPFH), represents a genetic Materials available but not contained in the kit: ing processes. failure of the mechanisms that turn off gamma chain synthesis ITEM CAT. NO. Sample Application at about four months after birth which results in a continued SPIFE 4000 Analyzer 1620, 1621 Electrophoresis high percentage of HbF. It is a more benign condition than the Gel Block Remover 1115 Predry true thalassemias and persons homozygous for HPFH have 10 AFSC Hemo Control 5331 Stain normal development, are asymptomatic and have no anemia. SPIFE 4000 White Sample Trays 2315 Destain The most common hemoglobin abnormalities: SPIFE 4000 Cassettes 1630 Dry Sickle Cell Trait SPIFE 4000 Maintenance Blotters 2307 Sample Application Applicator Load Time (mm:ss) 01:00 This is a heterozygous state showing HbA and HbS and a Applicator Load Speed 85 SPIFE 4000 Gel Staging Lid 2308 normal amount of HbA2 on cellulose acetate. Results on SPIFE 4000 Replacement Electrodes 1625 Application Rows 1 citrate agar show hemoglobins in the HbA and HbS migratory Materials needed but not provided: Row 1 Location (mm from gel edge): 55.0 positions (zones). 5% glacial acetic acid Apply Time (mm:ss) 01:00 Sickle Cell Anemia 0.85% saline Apply Cycles 1 This is a homozygous state showing almost exclusively HbS, Absorption Time (mm:ss) 00:00 STEP BY STEP METHOD although a small amount of HbF may also be present. Inter-Gel Start Delay (mm:ss) 22:00 I. SPIFE 4000 Preparation Sickle-C Disease Electrophoresis Voltage 160 1. Place SPIFE 4000 Applicator Blades (one per gel) begin- C S A F This is a heterozygous state demonstrating HbS and HbC. Minimum Current (mA) 10 Application Glycated ning in the first position at the top of the Applicator Tray. Point Sickle Cell-Thalassemia Disease Maximum Current (mA) 100 Hemoglobin 2. Fill the designated bottles with deionized water and destain. This condition shows HbA, HbF, HbS and HbA . Temperature (°C) 18 2 3. Add prepared acid blue stain to the appropriate stain bottle. In Sickle Cell β°-Thalassemia HbA is absent. Time (hh:mm:ss) 00:20:00 LIMITATIONS + The stain must be replaced after processing ten gels to avoid In Sickle Cell β -Thalassemia HbA is present in reduced Pre-Dry Temperature (°C) 62 contamination. Some abnormal hemoglobins have similar electrophoretic quantities. Time (hh:mm:ss) 00:10:00 4. Fill the DI Water Surfactant jar with deionized water and mobilities and must be differentiated by other methodologies. Thalassemia-C Disease Stain Stain Type Acid Blue replace the lid and tubing. Ensure that the ends of the Further testing required: This condition shows HbA, HbF and HbC. Absorption Time 00:04:00 tubing are below the water level. 1. Globin chain analysis (both acid and alkaline) and structural C Disease Destain Cycles 4 5. Remove the antisera/water reservoir from the antisera sta- studies may be necessary in order to positively identify some This is a homozygous state showing almost exclusively HbC. Time (hh:mm:ss) 00:02:00 tion. Lift the cover, fill the "H O" well and replace the cover. of the more rare hemoglobins. Thalassemia Major 2 Agitate Yes 6. Turn on the SPIFE 4000. Wait about 3 minutes after turn- 2. When a particular hemoglobin concentration varies signifi- This condition shows HbF, HbA and HbA . Dry Temperature (°C) 60 2 ing on the lower unit. Click the SPIFE 4000 icon on the cantly from the control, the migration will be affected. Time (hh:mm:ss) 00:11:00 screen for the instrument to initialize. Signs of Deterioration: The additive should be free of precipitate. 7. Using the prompts, prime the surfactant delivery system IV. Electrophoresis REFERENCE VALUES INSTRUMENT according to the instructions in the Operator’s Manual. 1. Click START on the screen and respond to the analyzer At birth, the majority of hemoglobin in the erythrocytes of the A SPIFE 4000 must be used to apply samples, electrophorese, II. Sample and Gel Preparation prompts. The analyzer will apply samples, electrophorese, normal individual is fetal hemoglobin, HbF. Some of the major stain, destain and dry the gel(s). stain, destain and dry the gels. Refer to the Operator’s Manual 1. Prepare hemolysates of patient specimens and controls as adult hemoglobin, HbA, and a small amount of HbA2 are also for detailed instructions. in­structed in the “Specimen Preparation”­ section. 2. The cassette with the gel will be dropped into the cas- present. At the end of the first year of life and through adult- SPECIMEN COLLECTION AND HANDLING 2. With the notch in the SPIFE 4000 White sample tray on the sette receptacle. hood, the major hemoglobin present is HbA with up to 3.5% 3. Remove the cassette(s) from the receptacle. If gel stor- Specimen: Whole blood collected in EDTA tubes is the spec- left, hand pipette 40 µL of patient or control hemolysate HbA2 and less than 2% HbF. imen of choice. into each sample cup in the top row. Load samples in age is required, remove and discard the two gel blocks. INTERPRETATION OF RESULTS Specimen Storage: If storage is necessary, whole blood and cups 1-7, skip the smaller cup in the center of the row Clean or wipe the non-gel side. If not, discard the used Most hemoglobin variants cause no discernible clinical symp- packed cells may be stored up to 1 week at 2 to 8°C. Frozen and then load samples in cups 8-14. gels, applicator blades and sample trays as biohazard- toms, so are of interest primarily to research scientists. Variants samples may produce an artifact band between HbF and HbA, 3. Stack the appropriate number of disposable sample trays, ous waste. are clinically important when their presence leads to sickling and band intensity may diminish, especially with hemoglobin C. with samples loaded, into the sample tray holder (one 4. Cassettes and carbon electrodes should be washed and disorders, thalassemia syndromes, life long cyanosis, hemolytic Specimen Preparation: Washed, packed cell hemolysates tray per gel), placing the first tray on the bottom. Place an dried after each use with deionized water. Refer to the anemias or erythrocytosis or if the heterozygote is of sufficient must be prepared for each patient specimen. additional empty sample tray on top of the stack to prevent Operator’s Manual for instructions. prevalence to warrant genetic counseling. The combinations A) Whole Blood sample evaporation of sample. Evaluation of the Hemoglobin Bands of HbSS, HbSD-Los Angeles and HbSO-Arab lead to serious 1. Centrifuge anticoagulated blood for 10 minutes to sepa- 4. Carefully open one end of the pouch, remove the gel from The hemoglobin gel should be inspected visually for the pres- sickling disorders.2 Several variants including HbH, E-Fort rate cells from plasma. the protective packaging and discard the overlay. ence of abnormal hemoglobin bands. Glycated hemoglobin Worth and Lepore cause a thalassemic blood picture.2 The two 2. Remove plasma. 5. Using a SPIFE 4000 Blotter C, gently blot the entire gel. migrates with HbF. The Helena AFSC Hemo Control provides variant hemoglobins of greatest importance in the U.S., in terms 3. Wash packed cells 3 times by resuspending in 5 to 10 Discard the blotter. a marker for band identification. of frequency and pathology, are HbS and HbC.2 Sickle cell volumes of normal saline solution (0.85% NaCl), centri- 6. Hold the gel so that the barcode is at the top. Place the gel Stability of End Product: The dried gels are stable for an anemia (HbSS) is a cruel and lethal disease. It first manifests fuging and aspirating supernatant. into the cassette by holding the gel backing in one hand indefinite period of time. itself at about 5 to 6 months of age. The clinical course presents 4. After washing the samples, prepare the samples by mix- and gently bending the gel. Slide each end of the gel back- Quality Control: The Helena AFSC Hemo Control (Cat. No. agonizing episodes of pain and temperature elevations with ing 10 µL sample to 100 µL Hemolysate Reagent. Vortex ing into the slots of the cassette to hold it in place. Align 5331) should be run on each SPIFE 4000 Acid Hemoglobin anemia, listlessness, lethargy and infarct in virtually all organs or shake vigorously for 15 seconds. the cutout in the gel backing with the alignment pin in the Gel. The control verifies all phases of the procedure and acts of the body. The individual with homozygous HbCC suffers B) Control cassette. as a marker to aid in the identification of the hemoglobins in mild hemolytic anemia which is attributed to the precipitation or AFSC (Cat. No. 5331) 1:2 (1 part control + 1 part Hemolysate 7. Ensure the gel blocks make good contact with the elec- the unknown samples. crystallization of HbC within the erythrocytes. Cases of HbSC Reagent) trodes to prevent skewed patterns. RESULTS disease are characterized by hemolytic anemia that is milder Figure 1 illustrates the electrophoretic mobility of bands on the PROCEDURE 8. Place the cassette with the gel into the humidor and cover than sickle-cell anemia. SPIFE 4000 Acid Hemoglobin Gel. Materials provided: The following materials needed for the the topmost cassette with the Gel Staging Lid. Close the The thalassemias are a group of hemoglobin disorders charac- procedure are contained in the SPIFE 4000 Acid Hemoglobin humidor lid to minimize gel dehydration. terized by hypochromia and microcytosis due to the diminished Kit (Cat. No. 2322). Individual items are not available. 9. Repeat Steps 4-8 for each gel needed. synthesis of one globin chain (the α or β) while synthesis of the 9,10 SPIFE 4000 Acid Hemoglobin Gels (10) NOTE: A maximum of 3 gels can be run at a time. other chain proceeds normally. This unbalanced synthesis Acid Blue Stain (1 vial) III. Electrophoresis Parameters results in unstable globin chains. These precipitate within the Hemolysate Reagent (25 mL) Using the instructions provided in the Operator's Manual, red cell, forming inclusion bodies that shorten the life span Citric Acid Destain (1 pkg) select of the cell. In α-thalassemias the α-chains are diminished or SPIFE 4000 Blotter C (10) -- Test Type: Acid Hb absent, and in the β-thalassemia the β-chains are affected. SPIFE 4000 Applicator Blades (10) Test Name: Acid Hb Another quantitative disorder of hemoglobin synthesis, hered- Destain Additive (28 mL) Check the programmed parameters for each of the follow- itary persistent fetal hemoglobin (HPFH), represents a genetic Materials available but not contained in the kit: ing processes. failure of the mechanisms that turn off gamma chain synthesis ITEM CAT. NO. Sample Application at about four months after birth which results in a continued SPIFE 4000 Analyzer 1620, 1621 Electrophoresis high percentage of HbF. It is a more benign condition than the Gel Block Remover 1115 Predry true thalassemias and persons homozygous for HPFH have 10 AFSC Hemo Control 5331 Stain normal development, are asymptomatic and have no anemia. SPIFE 4000 White Sample Trays 2315 Destain The most common hemoglobin abnormalities: SPIFE 4000 Cassettes 1630 Dry Sickle Cell Trait SPIFE 4000 Maintenance Blotters 2307 Sample Application Applicator Load Time (mm:ss) 01:00 This is a heterozygous state showing HbA and HbS and a Applicator Load Speed 85 SPIFE 4000 Gel Staging Lid 2308 normal amount of HbA2 on cellulose acetate. Results on SPIFE 4000 Replacement Electrodes 1625 Application Rows 1 citrate agar show hemoglobins in the HbA and HbS migratory Materials needed but not provided: Row 1 Location (mm from gel edge): 55.0 positions (zones). 5% glacial acetic acid Apply Time (mm:ss) 01:00 Sickle Cell Anemia 0.85% saline Apply Cycles 1 This is a homozygous state showing almost exclusively HbS, Absorption Time (mm:ss) 00:00 STEP BY STEP METHOD although a small amount of HbF may also be present. Inter-Gel Start Delay (mm:ss) 22:00 I. SPIFE 4000 Preparation Sickle-C Disease Electrophoresis Voltage 160 1. Place SPIFE 4000 Applicator Blades (one per gel) begin- C S A F This is a heterozygous state demonstrating HbS and HbC. Minimum Current (mA) 10 Application Glycated ning in the first position at the top of the Applicator Tray. Point Sickle Cell-Thalassemia Disease Maximum Current (mA) 100 Hemoglobin 2. Fill the designated bottles with deionized water and destain. This condition shows HbA, HbF, HbS and HbA . Temperature (°C) 18 2 3. Add prepared acid blue stain to the appropriate stain bottle. In Sickle Cell β°-Thalassemia HbA is absent. Time (hh:mm:ss) 00:20:00 LIMITATIONS + The stain must be replaced after processing ten gels to avoid In Sickle Cell β -Thalassemia HbA is present in reduced Pre-Dry Temperature (°C) 62 contamination. Some abnormal hemoglobins have similar electrophoretic quantities. Time (hh:mm:ss) 00:10:00 4. Fill the DI Water Surfactant jar with deionized water and mobilities and must be differentiated by other methodologies. Thalassemia-C Disease Stain Stain Type Acid Blue replace the lid and tubing. Ensure that the ends of the Further testing required: This condition shows HbA, HbF and HbC. Absorption Time 00:04:00 tubing are below the water level. 1. Globin chain analysis (both acid and alkaline) and structural C Disease Destain Cycles 4 5. Remove the antisera/water reservoir from the antisera sta- studies may be necessary in order to positively identify some This is a homozygous state showing almost exclusively HbC. Time (hh:mm:ss) 00:02:00 tion. Lift the cover, fill the "H O" well and replace the cover. of the more rare hemoglobins. Thalassemia Major 2 Agitate Yes 6. Turn on the SPIFE 4000. Wait about 3 minutes after turn- 2. When a particular hemoglobin concentration varies signifi- This condition shows HbF, HbA and HbA . Dry Temperature (°C) 60 2 ing on the lower unit. Click the SPIFE 4000 icon on the cantly from the control, the migration will be affected. Time (hh:mm:ss) 00:11:00 screen for the instrument to initialize. BIBLIOGRAPHY ® 1. Wintrobe, Maxwell M., Clinical Hematology, 6th Edition, SPIFE 4000 Acid Hemoglobin LeFebiger, Philadelphia, 1967. 2. Fairbanks, V.F., Diagnostic Medicine, Nov/Dec., 53-58, Cat. No. 2322 Procedure 1980. 3. Schneider, R.G., et al., Laboratory Identification of the Hemoglobins, Lab Management, August, 29-43, 1981. The SPIFE 4000 Acid Hemoglobin method is intended for the Storage and Stability: The gels should be stored horizontal- 4. Center for Disease Control, Laboratory Methods for qualitative determination of hemoglobins using agar in acidic ly at room temperature (15 to 30°C) and are stable until the Detecting Hemoglobinopathies, U.S. Department of Health buffer on the SPIFE 4000 system. expiration date indicated on the package. The gels must be and Human Services/Public Health Service, 1984. SUMMARY stored in the protective packaging in which they are shipped. 5. Schneider, R.G., Methods for Detection of Hemoglobin Hemoglobins (Hb) are a group of proteins whose chief func- DO NOT REFRIGERATE OR FREEZE THE GELS. Variants and Hemoglobinopathies in the Routine Clinical tions are to transport oxygen from the lungs to the tissues and Signs of Deterioration: Any of the following conditions may Laboratory, CRC Critical Reviews in Clinical Laboratory carbon dioxide in the reverse direction. They are composed of indicate deterioration of the gel: (1) crystalline appearance Sciences, 1978. polypeptide chains, called globin, and iron protoporphyrin heme indicating the agarose has been frozen, (2) cracking and 6. Schneider, R.G., et al., Abnormal Hemoglobins in a Quarter groups. A specific sequence of amino acids constitutes each peeling indicating drying of the agarose, (3) bacterial growth Million People, Blood, 48(5):629-637, 1976. of four polypeptide chains. Each normal hemoglobin molecule indicating contamination, (4) thinning of the gel blocks. 7. Huisman, T.H.J. and Schroeder, W.A., New Aspects of the contains one pair of alpha and one pair of non-alpha chains. 2. Acid Blue Stain Structure, Function, and Synthesis of Hemoglobins, CRC The non-alpha chains of fetal hemoglobin are called gamma. Ingredients: When dissolved as directed, the stain contains Press, Cleveland, 1971. 0.5% (w/v) acid blue stain. A minor (3%) hemoglobin fraction called HbA2 contains alpha 8. Schmidt, R.M., et al, The Detection of Hemoglobinopathies, and delta chains. Two other chains are formed in the embryo. WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO CRC Press, Cleveland 1974. The major hemoglobin in the erythrocytes of the normal adult NOT INGEST. 9. Weatherall, D.J. and Clegg, J.B., The Thalassaemia Preparation for Use: Dissolve the dry stain (entire contents is HbA, but there are small amounts of HbA2 and HbF. In Syndromes, Blackwell Scientific Publications, Oxford, 1972. addition, over 400 mutant hemoglobins are now known, some of vial) in 1 L of 5% glacial acetic acid. Mix thoroughly for 30 10. Lehman, H. and Huntsman, R.G., Man’s Haemoglobins, of which may cause serious clinical effects, especially in the minutes. J.B. Lippincott Co., Philadelphia, 1974. homozygous state or in combination with another abnormal Storage and Stability: The dry stain should be stored at 15 hemoglobin. Wintrobe1 divides the abnormalities of hemoglobin to 30°C and is stable until the expiration date indicated on the For Sales, Technical and Order Information and synthesis into three groups: package. The diluted stain is stable one year when stored at Service Assistance, call 800-231-5663 toll free. (1) Production of an abnormal protein molecule (e.g. sickle cell 15 to 30°C. anemia) Signs of Deterioration: The diluted stain should be a homo- Helena Laboratories warrants its products to meet our published specifications (2) Reduction in the amount of normal protein synthesis (e.g. geneous mixture free of precipitate. Discard if precipitate and to be free from defects in materials and workmanship. Helena’s liability under thalassemia forms. The stain must be replaced after processing ten gels this contract or otherwise shall be limited to replacement or refund of any amount to avoid contamination. not to exceed the purchase price attributable to the goods as to which such claim (3) Developmental anomalies (e.g. hereditary persistence of is made. These alternatives shall be buyer’s exclusive remedies. fetal hemoglobin (HPFH) 3. Hemolysate Reagent In no case will Helena Laboratories be liable for consequential damages even if The two mutant hemoglobins most commonly seen in the Ingredients: The reagent contains deionized water with 0.005 Helena has been advised as to the possibility of such damages. United States are HbS and HbC. Hb Lepore, HbE, HbG- M EDTA, 0.175% saponin and 0.07% potassium cyanide. The foregoing warranties are in lieu of all warranties expressed or implied includ- ing, but not limited to, the implied warranties of merchantability and fitness for a Philadelphia, HbD-Los Angeles and HbO-Arab may be seen WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO particular purpose. less frequently.2 NOT PIPETTE BY MOUTH. The reagent contains potassium Electrophoresis is generally considered the best method for cyanide. separating and identifying hemoglobinopathies. The protocol Preparation for Use: The reagent is ready for use as pack- for hemoglobin electrophoresis involves stepwise use of two aged. systems.3-8 Storage and Stability: The reagent should be stored at room Initial electrophoresis is performed in alkaline buffers. Cellulose temperature (15 to 30°C) and is stable until the expiration acetate was the major support medium used because it date indicated on the vial. yields rapid separation of HbA, F, S and C and many other Signs of Deterioration: Discard if solution has precipates or mutants with minimal preparation time. However, because flocculent. of the electrophoretic similarity of many structurally different 4. Citric Acid Destain hemoglobins, the evaluation must be supplemented by citrate Ingredients: After dissolution, the destain contains 0.3% agar electrophoresis which measures a property other than (w/v) citric acid. electrical charge. WARNING: FOR IN-VITRO DIAGNOSTIC USE. DO NOT This method is based on the complex interactions of the INGEST - IRRITANT. hemoglobin with an acid electrophoretic buffer and the agar Preparation for Use: Pour 11 L of deionized water into the support. The SPIFE 4000 Acid Hemoglobin method is a simple Destain vat. Add the entire package of Destain. Mix well until procedure requiring minute quantities of hemolysate to provide completely dissolved. Pour the entire contents of the Destain a screening method for the presence of abnormal hemoglobins Additive bottle into the prepared Destain and mix. such as HbS, HbC and HbF. Storage and Stability: Store the Destain at 15 to 30°C. It is PRINCIPLE stable until the expiration date on the package. Very small samples of hemolysates prepared from washed, Signs of Deterioration: Discard if solution becomes cloudy. packed cells are automatically applied to the SPIFE 4000 5. Destain Additive Acid Hb gel. The hemoglobins in the sample are separated by Ingredients: The product is a wetting agent. electrophoresis using a citrate buffer and are stained with Acid WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. DO Blue Stain. NOT INGEST - IRRITANT. REAGENTS Preparation for Use: Pour the entire contents of the Destain 1. SPIFE 4000 Acid Hb Gel Additive bottle into 11 L of prepared Citric Acid Destain. Ingredients: Each gel contains agar in citrate buffer with Storage and Stability: The additive should be stored at 15 0.25% EDTA and thimerosal as a preservative. to 30°C and is stable until the expiration date indicated on the package. Beaumont, Texas USA 77704 Pro. 212 Preparation for Use: The gels are ready for use as packaged. 4/19(4) Shaded areas indicate that the text has been modified, added or deleted.