These materials were supported by CoAg PS001285 from the CDC.

Haematology Job Aids

Standard Operating Policy and Procedures PROCEDURE: Manual WBC Cell Count 1. PURPOSE: Manual cell counts are performed when a parameter is below the automated instruments linearity to verify a doubtful result flagged by the instrument or when smear findings dont agree with the automated result. Manual cell counts may be used as a primary reference method for WBC. 2. SPECIMEN REQUIREMENTS: EDTA whole blood 3. REAGENTS: Turks Fluid (lyses RBCs and stains nucleus of WBCs) 4. EQUIPMENT: Manual WBC counts are performed with the use of a haemocytometer and WBC unopettes or Thoma glass pipettes. A. Neubauer Haemocytometer i. Each haemocytometer side has a counting chamber (two). Each counting chamber is 3mm x 3mm (area of 9mm2). ii. Each chamber is divided into 9 squares: each square is 1mm x 1mm (area of 1mm2). iii. Each of the four corner squares are divided into 16 smaller squares. iv. The centre square (1 mm2) is divided into 25 smaller squares each bordered by double-ruled lines. a. Each of the 25 small squares in centre square is 1/5mm per side (area of 1/25 mm2). b. Each of these 25 small squares is further subdivided into 16 smaller squares. c. Divisions (smaller squares within squares) help counting navigation. The chamber is made so it is recessed and when a coverslip is placed over the counting chamber area, there is a depth of 0.1 mm.

v.

Counting Chamber (one side)

vi. vii.

Each chamber has a total area of 9 mm (3 mm x 3 mm). Each chamber has a total volume of 0.9 mm3. Chamber DIMENSIONS = 3 mm x 3 mm x 0.1mm.

American Society for Clinical Pathology October 2010

5. 6. 7. 8. 9.

When cells are numerous, the cell dilution is big and the area counted is small (i.e. RBC's); fewer cells require a lesser dilution and a greater number of squares are counted (i.e. WBCs). SUPPLIES: Glass capillary tubes SPECIAL SAFETY PRECAUTIONS: N/A EQUIPMENT CALIBRATION AND MAINTENANCE: Microscope must be maintained to assure clean objectives and a clear image. Neubauer chambers need to be free of scratches with intact standardised coverslips. QUALITY CONTROL/ASSURANCE: Cell counts are performed in duplicate using two pipettes and counting both sides of the haemocytometer. Additional squares should be counted if the cell count is low. PROCEDURE: i. MIX EDTA blood sample well before obtaining aliquot of blood. Tilt to mix. iii. Fill 2 WBC Thoma pipettes to the 0.5 mark with blood by capillary action. There must be NO BUBBLES IN PIPETTES and excess blood must be wiped from outside of pipette. iv. Pipette WBC diluent to the top mark on the pipettes. Place pipettes on pipette shaker for at least 2 minutes. v. CLEAN chambers with alcohol and dry well; place coverslip on haemocytometer. vi. Invert pipette several times to mix blood with diluent before plating on haemocytometer chamber. Clean haemocytometer and put on coverslip. vii. Discard a few drops from the pipette and plate one dilution on each side of haemocytometer. Fill chamber smoothly and dont overfill; there should be no bubbles. viii. Allow cells to settle for 3 minutes in a petri dish with a damp cotton ball before counting. v. Use 10x (low power) objective and LOW light. ix. Focus on chamber groove when focusing, then move in to counting area; check cell distribution for even distribution in chamber squares before counting. x. Count all WBCs in the four large corner squares on both sides of the haemocytometer; include in your count white cells which touch the top and left boundary lines; exclude WBCs touching bottom and right lines.

viii.

xi. xii. xiii.

Use counter when counting cells; record # of cells counted for each chamber side on manual chamber count worksheets. The # of cells counted between sides must agree +20% to accept. If replating for a third count, first remix unopette, then discard 1 drop and plate. Correct for dilution and area counted (varies). STANDARD FORMULA: cells/cm = WBC counted (both sides) x dil x depth mm area counted sqmm (both sides) OR cells/mm3 = cells counted (both sides) x dilution factor x depth mm

American Society for Clinical Pathology October 2010

area counted sqmm (both sides) Report WBC counts to nearest hundred or nearest tenth, depending on units.
B.

MANUAL Red Cell Counts: PROCEDURE: Follow the procedure for white cell counting, the dilution is the same. The only difference is the counting area. For platelets the entire central area of the Neubauer chamber, all 25 squares are counted under a 40x objective. Both sides are tallied and then an average number of platelets is applied to the standard formula. The area is 1 mm. If you have a phase objective under your microscope, platelet is much easier as platelets are much more easily identified under phase contrast. Without phase contrast, platelets are much smaller than white cells, exhibit motion, and have a green sheen.

Counting chamber (one side)

10.

CALCULATIONS: FORMULA (to obtain # of cells/mm3 (cmm): A. Must correct for dilution used and area (squares) counted. B. Standard formula: cells/cmm = cells counted (both sides) x dilution factor___ depth mm x area counted sqmm (both sides) OR cells/mm3 = cells counted (both sides) x dilution factor x depth mm area counted sqmm (both sides) i. Dilution factor - invert dilution used (if 1:20 use x 20). ii. Depth - 0.1 mm - use this to obtain # cells/cmm (a volume) rather than # cells/mm2 (an area). iii. Often see depth factor of "x10" in numerator obtained by inversion of denominator. 11. EXPECTED VALUES/ REFERENCE RANGE: N/A 12. INTERPRETATION/RESULTS: 13. METHOD LIMITATIONS: A. Failure to mix blood specimen before sampling. B. Using wrong pipette - CAREFUL! C. Improperly filled chamber - sides won't agree due to poor cell distribution. MUST repeat if sides are not within acceptable duplication. D. Wrong dilution or calculation; always recheck math and check for correct units. E. Haemocytometer was not cleaned well, sat too long and/or dried up. F. Counting artifact as cells, counting cells in wrong area, or wrong counting due to incorrect light adjustment. NOTE: If you focus on the chamber counting area and cannot see any cells, you probably have too much light. 14. PROCEDURE NOTES: 15. REFERENCES:

American Society for Clinical Pathology October 2010

16. RELATED DOCUMENTS: N/A 17. AUTHOR: 18. APPROVAL SIGNATURES: Laboratory Director

Standard Operating Policy and Procedures

PROCEDURE: HEMOCUEPHOTOMETERFORHAEMOGLOBINMEASUREMENT 1. PURPOSE: The haemoglobin concentration in a fresh capillary or anticoagulated blood sample (EDTA preferred) is determined photometrically using a dry reagent system. The red cells are lysed and haemoglobin is converted to azidemethaemoglobin by sodium nitrite and sodium azide. This method of HGB measurement is a widely used point-ofcare test. SPECIMEN REQUIREMENTS: Capillary Blood or EDTA Whole Blood REAGENTS: Insert EQUIPMENT: HemoCue photometer SUPPLIES: HemoCue cuvette, puncture device, alcohol, cotton. SPECIAL SAFETY PRECAUTIONS: N/A EQUIPMENT CALIBRATION AND MAINTENANCE: Perform an instrument calibration check prior to use. The red control cuvette should read within + 0.3 gm/dl of the assigned value for the specific instrument to assure proper function. Document the calibration check result on the proper instrument sheet. QUALITY CONTROL: A haemoglobin normal control sample must be run once per day. The control must read within the assayed range to run the HemoCue for patient haemoglobins. Check that the HGB control value obtained reads on the QC chart and document the control result on the daily lab sheet. Repeat the control if not within established control limits. PROCEDURE: A. HemoCue Operation: i. Turn the HemoCue on using the switch in the back. The display screen should read "Hb". Pull the black cuvette holder out to the insertion position. After about 6 seconds, the screen should read "READY". ii. Place a cuvette (control cuvette or unknown) into the holder and insert to the "measuring" position. The HGB results in g/dl will be displayed in 45-60 seconds (results remain on screen ~5 minutes). B. Collection of capillary specimen (fingerstick): i. Equipment needed - HemoCue cuvette, puncture device, alcohol, cotton, etc. ii. On children and adults use the fingertip.....use of the middle finger is recommended. The finger should be straight but not tense; gently "milking" the fingertip stimulates blood flow. iii. Cleanse area with alcohol and wipe dry with cotton. While holding the finger, the puncture is made in a quick motion in the area just to the side of the fingertip but not close to the nail. iv. Using dry cotton, wipe away the first two to three drops of blood as they contain tissue fluid which could dilute specimen. Only slight pressure should be applied to the finger....avoid squeezing. Make sure that the drop of blood is big enough to fill the cuvette completely and without bubbles. C. Haemoglobin Measurement: i. Fill the cuvette with blood in a CONTINUOUS process WITHOUT BUBBLES. The cuvette needs to be filled all at once because the chemical reaction

2. 3. 4. 5. 6.

7.

8.

9.

American Society for Clinical Pathology October 2010

10. 11. 12. 13. 14. 15. 16. 17.

starts immediately and any delay in filling the cuvette results in incomplete red cell lysis. ii. Air bubbles in the centre of the cuvette require repeating the assay. iii. Wipe any excess blood from the outside of the cuvette, being careful not to touch the curved edge...important. iv. Place a cuvette (control or unknown) into the holder and insert to the "measuring" position. The results in g/dLwill be displayed in 45-60 seconds. Record results to the nearest tenth on result form. CALCULATIONS: N/A EXPECTED VALUES/ REFERENCE RANGE: INTERPRETATION/RESULTS: Check that your patient HGB and HCT results correlate.....i.e., does the HGB x 3 = HCT + 3%? H&H values obtained on the control sample will NOT correlate. METHOD LIMITATIONS: PROCEDURE NOTES: Filled cuvettes must be read within ten (10) minutes of filling to prevent alteration of the result caused by drying or specimen collection errors. REFERENCES: AUTHOR: APPROVAL SIGNATURE: Laboratory Director

Standard Operating Policy and Procedure:

PROCEDURE: MANUAL SPUN HAEMATOCRIT (HCT)/Packed Cell Volume (PCV) 1. 2. 3. 4. 5. 6. 7. 8. PURPOSE: The haematocrit measures the volume of packed red cells in a given volume of whole blood. SPECIMEN REQUIREMENTS: Capillary or EDTA Whole Blood REAGENTS: N/A EQUIPMENT: Microhaematocrit reader; microhaematocrit centrifuge SUPPLIES: Non-heparinised (EDTA sample) or heparinised (fingerstick sample) haematocrit/capillary plastic tubes; clay pads SPECIAL SAFETY PRECAUTIONS: Plastic capillary tubes are preferred over glass to avoid glass puncture with glass capillary tubes when inserting clay. EQUIPMENT CALIBRATION AND MAINTENANCE: (Insert centrifuge rpm checks and checks for maximum packing) QUALITY CONTROL: A. Duplicate spun HCT tubes must agree +2% to accept results or another HCT tube must be centrifuged. B. Controls must be within +2 standard deviations of the assayed control mean to accept patient HCT results. Check that HCT control values are within the acceptable range given on the QC chart. If the control does not "read", must repeat; use of a different control vial is suggested. C. Average the duplicate patient HCT results IF the control is acceptable and duplicate HCT tubes agree. PROCEDURE: A. MIX EDTA blood specimen and control vial well before taking blood sample. TILT to mix do NOT shake. B. Fill TWO microhaematocrit tubes 2/3 full for each EDTA sample and/or control.

9.

American Society for Clinical Pathology October 2010

C. Insert clay into the dry end of each tube, put tubes in holes in sheet of paper, and label the sheet with patient name. D. Balance tubes in centrifuge, PUT ON LID, and centrifuge 5 mins. Record centrifuge # and groove #'s used on result sheet. E. After centrifugation, use HCT reader to set 0 and 100, then read and record the HCT% obtained for each tube on the result sheet. Be sure to read only the red cell layer and exclude the buffy coat layer. Report manual haematocrit results to the nearest 0.5%. Refer to the diagram below of layers present in a spun haematocrit tube.

10. 11. 12. 13. 14. 15. 16. 17.

CALCULATIONS: N/A EXPECTED VALUES/ REFERENCE RANGE: INTERPRETATION/RESULTS: Check that your patient HGB and HCT results correlate HGB x 3 = HCT + 3%. H&H values obtained on the control will NOT correlate. METHOD LIMITATIONS: Sources of error include collection errors (i.e., clotted sample or EDTA tube less than half full), inadequate mixing or centrifugation, poor duplication of results, or improper use of the HCT reader (i.e., including buffy coat in HCT reading). PROCEDURE NOTES: Acceptable alternatives to clay include ________________ REFERENCES: AUTHOR: APPROVAL SIGNATURES: Laboratory Director

Standard Operating Policy and Procedures

PROCEDURE: HAEMOGLOBIN MEASUREMENT BY SPECTROPHOTOMETER 1. PURPOSE: Cyanmethaemoglobin method is the reference method for haemoglobin measurement. Haemoglobin can be measured manually using a spectrophotometer. Drabkins cyanmethaemoglobin reagent causes red cell lysis, release of haemoglobin and conversion of haemoglobin to a stable pigment called cyanmethaemoglobin. The absorbance (optical density or O.D.) of cyanmethaemoglobin at 540 nm is directly proportional to the concentration of haemoglobin present in the blood. (Some methods call for reading of % transmittance which is plotted on semi-log graph paper and is inversely proportional to the concentration.) All clinically significant haemoglobin forms are converted and measured by this method. EDTA whole blood is diluted in Drabkins reagent (contains NaHCO3, KCN, and KFeCN). A standard curve is prepared using solutions of known haemoglobin concentrations. Released Hgb + ferricyanide methaemoglobin KCN

American Society for Clinical Pathology October 2010

(Fe++)

oxidised

(Fe+++)

cyanmethaemogloin (stable pigment) Read at 540 nm.

2. 3.

4. 5. 6. 7.

SPECIMEN REQUIREMENTS: REAGENTS: Drabkins reagent: Potassium ferricyanide: K3Fe(CN)6 0.2 gm Potassium cyanide: KCN 0.05 gm Dihydrogen potassium phosphate (anhydrous): KH2PO4 0.14 gm Non-ionic detergent (enhances lysis of RBCs &decreases turbidity of solution) 0.50 mL Distilled water to 1,000.00 mL Once prepared, Drabkins solution must be kept in a dark bottle at room temperature and prepared fresh at least once a month to obtain quality patient results for haemoglobin. EQUIPMENT: Spectrophotometer, Sahli pipettes, test tubes SUPPLIES: SPECIAL SAFETY PRECAUTIONS: EQUIPMENT CALIBRATION AND MAINTENANCE: To prepare haemoglobin standard curve: A. A cyanmethaemoglobin standard vial (purchased) is opened just prior to preparing the standard linear graph for haemoglobin. B. Absorbance readings are made of the fresh standard and of dilutions of this standard diluted by Drabkins reagent (1:2; 1:3; 1:4) against a reagent blank set at 0 absorbance in a spectrophotometer at a wavelength of 540 nm. C. These absorbance readings are plotted on linear graph paper against haemoglobin concentrations. The points should present a straight line which passes through the point of origin.

Setting wavelength on spectrophotometers: 1. Select wavelength using wavelength control 2. Set the mode to Transmittance 3. With sample compartment empty and cover closed, adjust transmittance to zero or 0%T

American Society for Clinical Pathology October 2010

8. 9.

Select the Absorbance mode Insert a reference blank into the sample compartment and set 100%T or 0.0A 6. Insert unknown sample into the sample compartment and read measurement from display in percent transmittance or absorbance QUALITY CONTROL: 2 Levels of control are diluted as for EDTA whole blood PROCEDURE: A. 5 mL of Drabkins solution is added to each of the following tubes (12 x 75 mm) labelled accordingly:

4. 5.

i. reagent blank ii. haemoglobin normal control iii. haemoglobin abnormal control iv. patient (s) B. Add 20 L of normal control to tube labelled ii. C. Add 20 L of abnormal control to tube labelled iii. D. Add 20 L of each patients well-mixed blood to the appropriately labelled tube iv. E. Mix well. F. Allow to stand at room temperature for a minimum of 3 minutes (up to 15 min). Verify that the mixture is clear before reading. G. Transfer the mixture to a cuvette and read in a spectrophotometer. H. The absorbance (O.D.) is read against the reagent blank (set at 0.0) at 540 nm. I. The concentration of each control/specimen is determined from the standard curve graph set up earlier. J. If the concentrations of the controls are within the required limits for quality control, the patients results can be reported. 10. CALCULATIONS: N/A 11. EXPECTED VALUES/ REFERENCE RANGE: 12. INTERPRETATION/RESULTS: The cyanmethaemoglobin reagent blank should have an O.D. reading of 0.0 when measured against a water blank at 540 nm. 13. METHOD LIMITATIONS: Interferences include: Lipemia High WBC Abnormal haemoglobins (Hgb S and Hgb C) Abnormal proteins Anything causing turbidity or cloudiness 14. PROCEDURE NOTES: Haemoglobin results may not be reported if significant lipemia or very high WBC is present as solution is not clear. Haemoglobin results will be falsely elevated in these cases and will not match the Haematocrit. 15. REFERENCES: Sources: Essentials for the Small Laboratory and Physicians Office by Kathleen BecanMcBride, Doris L. Ross; Year Book Medical Publishers, Inc. Chicago, London, boca Raton, 1988. Hematology: Principles and Procedures, Fifth Edition by Barbara A. Brown, Lea & Febiger, Philadelphia, 1988. 16. AUTHOR: 17. APPROVED SIGNATURES:

American Society for Clinical Pathology October 2010

Standard Operating Policy and Procedures

PROCEDURE: PH METER, ORION - 420A PLUS 1. PURPOSE: A. The pH measurement system consists of a measuring electrode capable of detecting hydrogen ions, as well as the reference electrode. The pH electrode is a glass electrode, which develops a potential difference when the pH of the sample differs from the pH of the electrode fill solution. B. The reference electrode is a silver/silver chloride electrode filled with saturated KCl, separated from the sample by a cellulose membrane. A diffusion potential across the cellulose reference membrane is created between sample and saturated KCl in the reference electrode. This potential does not vary with the sample providing a constant potential for the electrochemical cell. The potential difference that develops between the measuring electrodes and the reference electrode varies with the ion activity in the sample. C. This Triode pH electode also has a built in thermistor for ATC (Automatic Temperature Correction) to 25C. SPECIMEN: A. Specimen must be aqueous. If the specimen is mucoid or has sediment in it, then centrifuge specimen before analysis. B. 2.0 ml is the minimum volume. The conical plastic tubes used for urine analysis work well for minimal volume specimens. REAGENTS:

2.

3.

A. B. C. D. E. F. G. H. I.
4. 5. 6. 7.

pH Electrode Storage Solution, 475 mL (Orion #910001) pH 4.0 Buffer Solution pH 7.0 Buffer Solution pH 10.0 Buffer Solution pH Cleaning solution A (Orion # 900021) pH Cleaning Solution B (Orion #900022) pH Cleaning Solution C (Orion #900023) pH Electrode Orion Triode Combination pH/ATC Probe for Orion A Series Meters. (Orion # 9157BN) DI water.

EQUIPMENT: SUPPLIES: SPECIAL SAFETY PRECAUTIONS: EQUIPMENT CALIBRATION AND MAINTENANCE: Calibration 2 Buffer calibration A. Remove the plug from the hole of the electrode and perform maintenance. B. Select 2 buffers. One should be the 7.0 and the other either the 10.0 or the 4.0 Buffer. Choose the one that will be closer to the range of the substance to be analysed. C. Press MODE until the pH mode indicator is displayed. D. Rinse electrodes first with distilled water and then the pH 7 buffer. E. Place the electrode in the 7.0 buffer. F. Press 2nd and cal to begin calibration. The date and time of the last calibration will be displayed.

American Society for Clinical Pathology October 2010

8.

9.

10. 11. 12. 13. 14. 15. 16. 17.

When READY is displayed next to the reading, indicating electrode stability, the reading will flash. Press yes. The buffer value is stored and meter display freezes for three seconds. The meter automatically switches to buffer two, indicated by the P2 on the display. H. Remove electrode from first buffer. Rinse with deionised water. I.Place electrode into second buffer. J. When READY is displayed beside the reading, indicating electrode stability, the reading will flash. Press yes. The buffer value is stored and meter display freezes for three seconds. K. After the second buffer value has been entered, press measure. The electrode slope will be displayed. SLP appears in the lower field while the actual electrode slope, in percent appears in the main field. A slope between 92% and 102% is acceptable. The meter automatically advances to the MEASURE mode. MEASURE is displayed above the main field. QUALITY CONTROL: A. 2 levels of QC are analysed with each run. B. Refer to the quality control products list for storage and handling instructions for the control materials. The worksheet will state which quality control to run. C. Acceptable: The run is acceptable when both controls are within the established range. D. Unacceptable: The run is unacceptable when one or more controls are not within the established range. Perform one or more of the following and document in Meditech under corrective action taken. i Repeat the Control. ii Repeat the fresh Control. iii Perform 2 Buffer Calibration. iv Clean electrode v Contact Lead Tech or Supervisor. vi Contact Manufacturer. PROCEDURE: A. After the above calibration procedure if performed; proceed with next step. B. Remove electrode from buffer. Rinse with deionised water. Place electrode into sample. When READY is displayed beside the reading. Record the sample results. C. Run all pH measurements in duplicate. D. Store electrode with the plug in the hole of the electrode and immerse tip in pH Electrode Storage Solution. CALCULATIONS: EXPECTED VALUES/ REFERENCE RANGE: INTERPRETATION/RESULTS: Report results to the nearest 0.1 METHOD LIMITATIONS: This Triode pH electrode should not be used in samples containing organic solvents. PROCEDURE NOTES: If persistent drifting, then perform electrode maintenance. See manual for list of error messages. REFERENCES: Orion A plus 420 Manual AUTHOR: APPROVAL SIGNATURES:

G.

American Society for Clinical Pathology October 2010

10

Centrifuge Maintenance Procedure Weekly or biweekly depending on usage: Clean interior components with soap and water followed by freshly made 10% v/v bleach solution, including sample buckets. Wearing protective gloves, wipe interior sides and bottom taking care when removing broken pieces of glass. Weekly: Place two equally balanced containers into the centrifuge, cover and operate at the most commonly used speed, listening for unusual vibrations. Check the braking mechanism to ensure a smooth gradual stop. Monthly: Inspect gasket and check for wear and defects and inspect cover latch for appropriate seal. Inspect head, head shaft and coupling for evidence of wear, cracks in fitting, corrosion, uneven wear and signs of fatigue. Inspect brushes for wear and replace according to manufacturers instructions. Monthly: Check the timer of the centrifuge at 15 minutes, 10 minutes, 5 minutes and 1 minute for the time the centrifuge motor is spinning (reaches the desired RPM until the motor shuts off) using a stopwatch. Monthly: Check the revolutions per minute at several commonly used speeds including 3000 and 1500 rpms while centrifuging a balanced load (after it has reached stable speed) using a tachometer aimed at the reflective strip viewed through the top of the centrifuge. Quarterly: Lubricate centrifuge shaft according to manufacturers instructions, if applicable. Centrifuge Maintenance Procedure Checklist Month___ Year_____ Weekly Check balance and brake Date and sign. Week 1 Week 2 Week 3 Week 4 Weekly Clean interior Date and sign. Monthly Inspect parts including brushes Date and sign. Monthly Timer check for 1, 5, 10, 15 minutes. List times Monthly RPM check for 1500, 3000. List RPMs Quarterly Lubrication if necessary, Date and Sign.

American Society for Clinical Pathology October 2010

11

PLATELET ESTIMATE Following are instructions to estimate platelet numbers from a smear: 1. In a thin area of the smear where only a few RBCs overlap, examine 5-10 different fields under oil immersion lens. 2. Determine the average number of platelets per field. Multiply this result by 20,000 to obtain an approximation of the platelet count. A normal platelet count should show 8 to 20 platelets per field. 3. Example: Average number of platelets per field=10. 4. 20,000 x10= 200,000/cu mm= smear estimation REFERENCE: Barbara A Brown, Hematology Principles and Procedures, 5th edition 1988 Lea & Febiger, Philadelphia, p. 101

WBC ESTIMATE The number of WBCs are estimated from the smear using the high power lens and in an area where the red cells are overlapping. 1. Count the # of WBCs in 10 HIGH (40x) power fields (HPFs); find average. 2. The average # WBCs per HPF x 2,000 = WBC estimate/cmm. 3. The estimate should agree 20% with the WBC count. ESTIMATE NOTES: Platelet and WBC estimates include those fields with no cells counted. It may be helpful to count cells in 5 thinner fields and 5 thicker fields. WBC estimates include smudge or broken cells. PLT/WBC estimates are used to check the validity of automated/manual cell counts; estimates are not reported.

American Society for Clinical Pathology October 2010

12

Standard Operating Policy and Procedures

PROCEDURE: SICKLE SCREEN 1. PRINCIPLE: The reaction using Sickle STAT is based on the relative insolubility of Haemoglobin S, when combined with a buffer and a reducing agent. When the Sickle STAT reagent powder is mixed with the Sickle STAT Buffer and blood sample is added according to the directions for testing, blood containing Haemoglobin S will form a cloudy, turbid suspension. Other haemoglobins are more soluble and will form a transparent solution when tested. The Sickle STATs sole purpose is to confirm the presence of Hb S. It is specific for this haemoglobin and will distinguish it from other variant haemoglobins that may mimic Hb S electrophoretically. The Screen is also useful in identifying the rare variants with two amino acid substitutions, one of which is the sickle mutation (Hb SC Harlem, C Ziguinchor, S Antilles, and S Travis). Although these haemoglobins sickle, their electrophoretic patterns are not those of typical sickle cell patients and the lab results and clinical features may not correlate. NOTE: Sickle STAT identifies only the presence of Hb S, not its quantity. Therefore, this method does not easily distinguish between sickle cell trait and sickle cell anaemia. Furthermore, the test does not detect the presence of other common haemoglobin anomalies, such as Hb C, Hb E, or beta-thalassemia. SPECIMEN REQUIREMENTS: A. 50 L of pipettable anticoagulated blood: 1. EDTA is preferred anticoagulant. 2. Heparin, sodium or potassium oxalate, sodium citrate, ACD, CPD, or CPD1 may also be used. B. Blood can be used up to two weeks for sickle screen (if refrigerated). C. Sickle screens are not recommended on children under 3 months of age since the level of Hb S may be below detectable levels for the test. False negatives may occur up to one year of age. REAGENTS: CHEMBIO DIAGNOSTICS SICKLE-STAT TEST KIT A. Phosphate buffer with stabiliser. B. Pre-filled reaction tubes containing reducing agent (sodium dithionite). C. All reagents are stable until expiration date on label. EQUIPMENT: SUPPLIES: SPECIAL SAFETY PRECAUTIONS: N/A EQUIPMENT CALIBRATION: N/A QUALITY CONTROL: A. A positive control, blood with known Hb S, is tested with each patient run. B. If patient result is positive, it is necessary to test a known Hb S negative control. C. EDTA blood of known Hb S positive patients is saved in refrigerator and is used as a positive control. D. EDTA blood from a Hb S negative patient can be used as a negative control (blood bank donor). PROCEDURE: A. Remove SICKLE-STAT buffer from refrigerator. B. Remove as many tubes as needed from kit. Place tubes in Sickle-Stat observation rack.

2.

3.

4. 5. 6. 7. 8.

9.

American Society for Clinical Pathology October 2010

13

10. 11. 12.

13.

14. 15.

C. Squeeze buffer to fill line on tube (4ml). Mix and allow to come to room temperature. D. Add 50 L of patients blood to one 4 cc tube and 50 L of positive control blood to a second 4 cc tube. (If the patient is positive then it will be necessary to run a negative control.) E. Cover tubes with cap and invert several times to ensure proper mixing. F. Wait six minutes and examine each tube in the Sickle-STAT observation rack. CALCULATIONS: N/A EXPECTED VALUE: INTERPRETATION: Haemoglobin S (Hb S) is an inherited characteristic, which occurs either in the homozygous (S/S-Sickle Cell Anemia) or heterozygous (A/S Sickle Cell Trait) state. Hb S may also be found with other abnormal haemoglobins or with thalassemia. Hb S tends to form tactoids or liquid crystals within the erythrocytes under conditions of low oxygen tension resulting in the characteristic sickle shape distortion of the red cell. Sickle Cell disease, in the homozygous state, is often fatal before adolescence; but with medical management, survival for forty years or longer is possible. The most common clinical symptom is severe haemolytic anemia with vascular occlusions involving the spleen, kidneys, lungs, retinas, central nervous system, and bones. Refractory ulcers of the lower legs and episodes of severe back, abdominal or joint pain are frequent. Even though individuals with sickle trait are usually asymptomatic, in certain circumstances of reduced oxygen tension (during anaesthesia, flights in poorly pressurised airplanes and severe pneumonia), these individuals may experience difficulties. REPORTING RESULTS: A. Report controls and patient results using the RETIC worksheet. B. Report positive or negative only. C. A positive test for Hb S is indicated by a cloudy supernatant through which the ruled black lines are not clearly observable. D. A negative test is indicated by a nearly transparent suspension. This is indicated by the ability to clearly see the ruled lines on the back of the observation rack. E. In reading the results of patient samples it is acceptable to have a very small amount of turbidity/cloudiness present in a negative sample due to the proteins and debris. Any true positives, especially those with a total haemoglobin concentration in the normal range (~11-14 g/dL) will present a much more distinct turbidity and will obscure the lines on the rack much like the positive control. F. Coarse flocculation in the reaction tube may occur if abnormal concentrations of serum proteins are present. When this occurs, the patients specimen should be washed once with normal saline and centrifuged. The supernatant is then decanted and the cells are re-suspended with normal saline to a normal haemoglobin concentration. This sample can now be tested. REFERENCE RANGE: Normal = Negative METHOD LIMITATIONS A. Negative screens on patients under one year should be repeated after one year of age. B. Low levels of Hb S are frequently observed in infants below three months of age. Screens should be performed after three months C. Sickle screen is a qualitative test and does not distinguish between Sickle Cell Disease (S/S) and Sickle Cell Trait (A/S). All positive results should be further evaluated by haemoglobin electrophoresis. Cases of Hb C trait and beta-thalassemia trait (also common in African Americans - together they are nearly half as frequent as Hb S trait and should be recognised) will not be detected by this test. Therefore,

American Society for Clinical Pathology October 2010

14

16. 17.

18. 19.

specimens that are negative by sickle stat may still need Hgb Eletrophoresis if clinically warranted. PROCEDURE NOTES: Haemoglobin levels under 5.0 grams can cause a false negative result. Double the sample volume in these instances. REFERENCES: Lehmann, Saunders Manual of Clinical Laboratory Science, pg. 893-895, 1998. Lee, Bithell, etc, Wintrobes Clinical Hematology, pg 1068-1084, 1993. Steine-Martin, et al, Clinical Hematology, pg. 95-96, 196-197, 201-203, 1998. Chembio diagnostics SICKLE-STAT TEST KIT package insert. AUTHOR: APPROVAL SIGNATURES: Laboratory Director

Standard Operating Policy and Procedures

PROCEDURE: MANUAL BLOOD SMEAR PREPARATION 1. PURPOSE: Examination of the well prepared and well-stained blood smear is one of the most efficient and important initial laboratory tests in the evaluation of a patient with an abnormal blood count. In addition, in some instances, despite a normal blood count, the blood smear can be very helpful in the diagnosis and follow-up of many haematologic, infectious, inflammatory, metabolic, and immunologic disorders. Examination of a peripheral blood smear for red blood cell morphology and number, appearance of platelets, and performance of a leukocyte differential count is routinely used. The correct interpretation of changes in any of the blood components on a peripheral blood smear depends on the quality of the blood smear and its staining REAGENTS: Romanowsky type stain such as Wright-Giemsa; buffer solution EQUIPMENT: Microscope SUPPLIES: New, clean 25 mm x 75 mm X 1 mm, glass microscope slides of good quality that are pre-cleaned before use. The slides should be of high quality glass and have smooth ground edges. Applicator sticks/or glass rod /pipette SPECIMEN: Whole blood (lavender top tube with EDTA anticoagulant) The EDTA samples should be stored at room temperature (18 - 25o C) and should not be refrigerated. IMPORTANT: Causes for rejection: clotted specimen. SPECIAL SAFETY PRECAUTIONS: N/A EQUIPMENT CALIBRATION AND MAINTENANCE: Microscope objectives must be clean and well maintained. QUALITY CONTROL: The push-wedge blood smear should have the following appearance: A. The smear should cover approximately two-thirds of the length of the slide (minimum of 2 inches). B. It should be narrower than the slide with smooth margins so cells at edges may be examined. C. The spread should be smooth with gradual transition from thick to thin areas. (There should be 10 or more low power fields in which the red cells barely touch but don't overlap, and the white cells should be evenly distributed.) D. The smear should be free of holes, scratches, ridges, grease and dirt contaminants. E. Its colour should be reddish brown to orange-brown. PROCEDURE: The push or wedge type of smear continues to be the most prevalent technique and the easiest to master.

2. 3. 4. 5. 6. 7. 8.

9.

American Society for Clinical Pathology October 2010

15

A.

10. 11. 12. 13.

14.

Place a small to medium (approximately 2-mm diameter) drop of capillary needle blood or well-mixed EDTA anticoagulated blood in the centre of a 1" by 3" clean glass slide, about 2" in from the end of a clear slide with a" from the frosted area of a frosted slide. The slide should be on a flat surface and held fixed by one or two fingers of one hand. Use the applicator stick or glass rod/pipette to make a drop. B. Hold spreader slide between the thumb and middle finger of the other hand with the index finger at the top centre to provide slight weight and stability. C. Completely back the spreader slide into the blood drop, which will run along the rear edge to the side edges of the spread slide. D. Hold the spreader slide at a 30-degree angle for best distribution. E. When the blood reaches the side edges, quickly move the spreader slide to the opposite end with an even motion. F. Allow the smear to air dry. Do not blow on it. INTERPRETATION/RESULTS: Perform manual differential (See procedure). CALCULATIONS: N/A EXPECTED VALUES/ REFERENCE RANGE: N/A METHOD LIMITATIONS: A. Excessive amounts of EDTA will create artifacts that make red blood cell morphology difficult to interpret. The artifacts of red blood cell morphology seen in blood smears prepared from EDTA anticoagulated blood include the following: i. Spiculated red blood cells ii. Target cells iii. Rouleaux iv. Spherocytes v. Stomatocytes vi. Punched-out red blood cells B. In general, the longer that blood is exposed to EDTA in an evacuated tube, the more marked will be the artifactual changes. PROCEDURE NOTES: A. Failure to keep the spreader slide at a 30o angle with the flat slide results in the following: i. A higher angle produces a thick smear. ii. A lesser angle produces a thin smear. B. Speed of the spread affects the distribution. Rapid motion produces a thick smear; slow motion produces a thin, uneven spread. C. Anaemic blood should be spread with a faster motion or a greater spreader/slide angle. D. Polycythemia and newborns' blood should be spread more slowly or with a smaller spreader/slide angle. E. A bullet shaped blood film results from advancing the pusher slide before the drop of blood has spread uniformly behind the pusher slide. The straightedge blood film is preferred to the bullet-edge film because the leukocytes are distributed more uniformly. F. Good quality smears with good distribution of red cells in the examination area of the slide may also be difficult or impossible to prepare in patients with cold agglutinin disease, cryoglobulinemia, monoclonal gammopathies, or in the presence of some drugs such as miconazole. G. Blood films must be dried quickly to obtain good red blood cell morphology. If the blood film dried slowly in a humid environment, a "punched out" artifact of the red cells may result leading to an erroneous interpretation of hypochromia of the red cells.

American Society for Clinical Pathology October 2010

16

H. I.

Heat should never be used to dry blood films rapidly because it will cause shrinking of the blood cells and make their identification difficult. Common causes of a poor blood smear: i. Drop of blood too large or too small. ii. Spreader slide pushed across the slide in a jerky manner. iii.

15.

16. 17.

Failure to keep the entire edge of the spreader slide against the slide while making the smear. iv. Failure to keep the spreader at 30 degree angle with the slide. v. Failure to push the spreader blade completely across the slide. REFERENCES: Henry, J.B. Clinical Diagnosis & Management by Laboratory Methods, 20th Edition. WB Saunders, 2001 McKenzie, B.Sherlyn. Textbook of Hematology, 2nd edition. William and Wilkins, 1996 Diff-Safe Blood dispenser, current package insert. Alpha Scientific Corporation, Southeaster, Pa. PREPARED BY: Marian Cavagnaro, Memorial Regional Hospital APPROVAL: Laboratory Director

Standard Operating Policy and Procedures

PROCEDURE: SODIUMCITRATEFORPLATELETCLUMPING 1. PURPOSE: Ethelenediaminetetraacetic acid (EDTA) is the anticoagulant of choice when performing platelet counts or an evaluation of platelet morphology on a Wrightstained peripheral blood smear. However, EDTA may also induce nonspecific platelet clumping or platelet satellitosis (pseudothrombocytopenia). In platelet satellitosis, platelets cling to the outer edges of leukocytes. In both platelet clumping and platelet satellitosis, the total platelet count may be falsely decreased. A review of the stained blood smear using 10x or 40x objectives will confirm the presence of either platelet clumping or platelet satellitosis. To eliminate the clumping or satellitosis and obtain an accurate platelet count, a fresh specimen can be collected using sodium citrate as the anticoagulant. SPECIMEN REQUIREMENTS: Standard venipuncture specimen drawn into a sodium citrate tube. It is very important that a ratio of 9 parts blood to 1 part anticoagulant be maintained in the evacuated tubes with sodium citrate. Therefore, a completely full tube must be collected. REAGENTS: 3.2% sodium citrate (evacuated tubes with light blue stoppers) EQUIPMENT: SUPPLIES: Standard venipuncture equipment and supplies are used to recollect the specimens. SPECIAL SAFETY PRECAUTIONS: N/A EQUIPMENT CALIBRATION & MAINTENANCE: N/A QUALITY CONTROL: Standard quality control procedures used with automated analysers. PROCEDURE: a. Perform the platelet count on a sodium citrate specimen in the usual manner. b. Correct the platelet count for the dilutional effect of liquid citrate. c. Report the corrected platelet count.

2.

3. 4. 5. 6. 7. 8. 9.

American Society for Clinical Pathology October 2010

17

10.

CALCULATIONS: The platelet count results from the specimen recollected in sodium citrate must be corrected because of the dilution of the blood sample with the anticoagulant. When sodium citrate is used, the blood is actually 9/10 of the total volume. The dilution factor is the reciprocal of the dilution, or 10/9, which equals 1.1. Therefore, the new platelet count is multiplied by 1.1 to obtain the final result. The following example illustrates how to obtain an accurate platelet count on an EDTA sample that demonstrates platelet clumping. Original platelet count 8.0 x 109/L EDTA specimen Specimen recollected in sodium citrate and platelet count determined Platelet count from sodium citrate specimen corrected for dilutional effect of anticoagulant 160 x 109/L

160 x 109/L x 1.1 = 176 x 109/L

11. 12. 13.

15.

Platelet count to report 176 x 10 9/L 3 EXPECTED VALUE: 140,000-400,000/mm INTERPRETATION/RESULTS: METHOD LIMITATIONS: Platelet clumping has been documented to occur in anticoagulants other than EDTA, including oxalate and heparin. Furthermore, some cases of pseudothrombocytopenia have even been associated with the use of citrate. However, some studies suggest that platelet clumping may be time and temperature dependent. Therefore, if platelet clumping is suspected in a specimen collected in an anticoagulant other than EDTA, an accurate platelet count may be obtained by following these guidelines: a. Perform the platelet count immediately after collecting the specimen. b. Maintain the blood sample at 37o C. c. Collect a skin puncture sample without anticoagulant and determine a platelet count using a manual method. d. Attempt to disperse the platelet clump by using a vortex on the EDTA specimen and re-analysing the sample for platelets. REFERENCES: Maedel, LB and Brown, KA. Spurious Thrombocytopenia and Automated Blood Cell Analysis. ASCP Hematology Tech Sample H-3 (1998). McKenzie, S. Clinical Laboratory Hematology, Pearson Prentice Hall, 2004, p. 142.

Standard Operating Policy and Procedures

PROCEDURE: PATIENTCONTROLSONAUTOMATEDHAEMATOLOGYANALYSERS 1. 2. 3. 4. PURPOSE: As an alternative or supplement to using assayed quality controls for daily quality control, patient samples may be used to assess the precision and stability of the analyser on a daily basis. SPECIMEN REQUIREMENTS: EDTA Whole Blood REAGENTS: Not applicable EQUIPMENT: Automated Hematology Analyser

American Society for Clinical Pathology October 2010

18

5. 6. 7. 8. 9.

10. 11. 12. 13.

14. 15. 16. 17.

SUPPLIES: N/A SPECIAL SAFETY PRECAUTIONS: N/A EQUIPMENT CALIBRATION & MAINTENANCE: N/A QUALITY CONTROL: N/A PROCEDURE: a. Select at least two patient samples randomly daily with different analysed values (low, normal, and high) and refrigerate for a period not to exceed 24 hours. b. Carefully mix samples and run patient controls every 4 hours on the analyser and check reproducibility of results. c. Compare the results with originally assayed results. d. Control results must agree within the following established control limits: WBC +/- 1.1 RBC +/- 0.12 Hb +/- 0.40 Hct +/- 1.90 MCV +/- 3.0 MCH +/- 1.5 MCHC +/- 2.2 PLAT +/- 40.0 e. Print out all control values and maintain with the quality control records. f. If any result falls outside of expected ranges, repeat the specimen and then follow troubleshooting steps for out-of-control situations. g. Patient controls may be maintained in the refrigerator and used for a period of 24 hours. CALCULATIONS: N/A EXPECTED VALUE: N/A INTERPRETATION/RESULTS: N/A METHOD LIMITATIONS: Patient controls should be used to assess parameter reproducibility on a previously calibrated instrument. Patient controls may not be used to assess the accuracy of testing. Patient controls are ideally used in combination with assayed controls on a calibrated instrument. PROCEDURE NOTES: REFERENCES: Howard University Hospital Department of Pathology Standard Operating Procedures/Hematology. AUTHOR: APPROVAL: Laboratory Director

American Society for Clinical Pathology October 2010

19

EXAMPLE: Automated Haematology Analyser Control Log Date

Time

Haematology Controls Low Normal High

Lot#

Exp Date

Tech

Comment

EXAMPLE: Haematology Department Automated Haematology Analyser _____________ Quality Control Corrective Action Log

Instrument Serial# Date Performed Description Of QC Problem/Corrective Action Taken By

American Society for Clinical Pathology October 2010

20

American Society for Clinical Pathology October 2010

21

EXAMPLE: Haematology Department Automated Haematology Analyser _____________ Preventative Maintenance Log Instrument Serial#: Date Daily Maintenance StartUp 1 2 3 4 5 6 7 8 9 10 11 12 EXAMPLE: Haematology Department Automated Haematology Reagent Log Date Loaded Name of Reagent Lot# Expiration Date Date Opened Tech Initials ShutDown Tech Weekly PM Monthly PM As Needed PM / Corrective Maintenance

American Society for Clinical Pathology October 2010

22

American Society for Clinical Pathology October 2010

23

EXAMPLE: Haematology Department Communication Log Date Time Tech Problem/Message Action Taken Follow Up

American Society for Clinical Pathology

24

October 2010