Standard Operating Procedure

Transcription

1 Subject Differential: Counting and Morphology Index Number Lab-5074 Section Laboratory Subsection Hematology Automated Laboratory Category Departmental Contact Moldenhauer, Emily C Last Revised 11/28/2017 References Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers for Medicare and Medicaid Services (CMS) and/or COLA. Applicable To Employees of Gundersen Healthcare System laboratories, Gundersen St. Joseph s Health Services laboratories, Gundersen Tri-County Hospital laboratories, Gundersen Boscobel Area Hospital and Clinics laboratories, and Gundersen Palmer Lutheran Hospital and Clinics laboratories. Detail PRINCIPLE: To establish the absolute value of each type of leukocyte, confirm the electronic differential and describe RBC and Platelet morphology. Electronic Differential - The analyzer determines cell identification based on predetermined criteria set by the manufacturer. When criteria are exceeded or cell population is not defined, the instrument FLAGS the population of cells as positive, indicating that they should be reviewed visually by a tech. Scan The technologist scans the slide at least seconds and/or fields to determine presence of abnormal WBCs and/or to confirm the accuracy of the electronic differential. If in agreement and with no abnormalities, the differential is reported as Electronic diff verified by slide review. If not, a manual differential is reported. RBCs and platelets are examined and morphology is reported with every slide review. NOTE for SYSMEX KX-21N and Sysmex poch-100i users - If mixed cell result >11%, perform scan. If results of scan show an abnormal amount of Baso, Eos or Monos, perform a manual differential. If the manual differential shows that all Baso, Eos, and Monos are within normal limits, report electronic differential. If the Baso, Eos or Mono numbers are abnormal, report the manual differential. Manual differential - The first 100 leukocytes on a blood smear are identified and counted by a technologist using the manual counter, keyboard, or keyboard and Data Innovation s Middleware (DI); the leukocytes are differentiated and the cell types are reported as absolute values. CLINICAL SIGNIFICANCE: The leukocyte differential count has been one of the principal tests performed in the hematology lab. In addition to providing data as to the absolute number of each type of leukocyte present on the blood Page 1 of 13

2 smear, performance of the differential reassures the physician that someone has reviewed the smear for any cellular abnormalities. SPECIMEN: 1. Patient Preparation: N/A 2. Type of Specimen: Whole blood or capillary collected in EDTA. 3. Amount of Specimen: One drop (50 ul) of well mixed EDTA whole blood. 4. Collection and handling requirements: Blood should be mixed immediately upon collection in EDTA to ensure specimen does not clot. 5. Stability and Storage: Specimen can remain at room temperature for 4 hours, use with caution if over 4 hours. If refrigerated, let the tube sit at room temperature for 30 minutes and invert by hand, end to end, before preparing the smear. 6. Criteria for unacceptable specimens: a. unlabeled specimen b. Clotted specimen c. Heparinized specimen d. Sodium citrate tube may be used for differential and but not for any estimation since the dilution factor will affect result. e. Blood greater than 4 hours old should be used with caution, since the integrity of the WBCs will be in question. f. Tolerance Limits for an acceptable slide: i. Minimum of 2.5 cm in length and at least 1 cm from end. ii. Gradual termination in thickness, with feather edge at termination. iii. Acceptable morphology (without artifacts) within working area. iv. Film should be narrower than the slide and have smooth continuous side margins. v. Stain should be even and consistent with correct color for each structure (i.e. deep blue nucleus, pink or blue cytoplasm). vi. Leukocytes should be well preserved; beware of anticoagulant effects such as excessive vacuolization. vii. There should be less than 2% smudge cells (unless lymphoproliferative disorder). 7. Specimen must be labeled with at least two unique identifiers; date, name and Medical Record # is recommended. The SP-10 slide stainer will label slides with the date, name a barcode Sample ID # and a barcode readable by the Cellavision. The slide may be labeled with the barcode ID aliquot sticker containing the patient s name and barcode number. (Handwritten is acceptable). REAGENTS / MATERIALS: See procedure Lab-5137 Automated Stainer, SP-10-XN-9000, Lab-1537 Staining Blood Smears Using the Hematek and Lab-1536 Blood Smears, One Step Staining for reagent lists. EQUIPMENT / INSTRUMENTATION: 1. SP-10 will be used for making and staining blood slides at the La Crosse site. Refer to Lab-5137 Automated Stainer, SP-10-XN-9000 for proper use. 2. The HemaTek slide stainer if available, see Lab-1537 Staining Blood Smears Using the Hematek for proper use. 3. Diff Safe, plain Hct tubes, or the MINIPREP, Geometric Data, A SmithKline Beckman Co., Wayne, PA for making slides manually. Page 2 of 13

3 4. Light microscope, Nikon Eclipse E400, Nikon Eclipse 50i or Olympus. 5. Manual differential counter, computer keyboard or computer keyboard and Data Innovations Middleware (DI) for counting the differential. 6. When necessary, the La Crosse site will use the Hematek Stainer as a backup automated stainer. Refer to the Hematek work-aid attached to policy Lab-5137 Automated Stainer, SP10-XN Glass slides. Microscope Maintenance: Microscopes are cleaned a minimum of once/week by lab staff. A thorough cleaning is performed at least annually by professional microscope cleaning staff. QUALITY CONTROL: The quality of each slide is assessed for correct color of the inner structures. For example, in a neutrophil, the nucleus is deep blue-purple and cytoplasm is pinkish. If needed, another slide is made and stained. Proficiency testing: Testing of purchased survey specimens is rotated through technical staff. Implementation PROCEDURE: When using the Sysmex KX-21N or Sysmex poch-100i, slides are made and stained for all CBCs, hemograms with flags and abnormal platelets. On CBCs, slides will be scanned and a manual differential performed if the scan does not agree with the automated results. See Note below under Scan #4. When using the Sysmex XS-1000i, slides are made and stained on all Positive flagged CBCs. The criteria for Positive CBCs are: 1. All instrument flags as identified in our DI rules 2. Children 1 month of age with an ANC <2.0 When using the Sysmex XN-9000, Automated slides are made and stained by the SP-10 on all "Positive" flagged CBCs. The criteria for a "Positive" CBC are: 1. All instrument flags as identified in our DI rules 2. Children 1 month of age with an ANC < NRBC >5.0% and questionable separation in NRBC histogram 4. Immature Gran# >0.5 Preparation of Smear: 1. Manual Smear Preparation: a. Mix blood well before smear is made by inverting end to end 10 times. b. Prepare a manually made slide with the MINIPREP or by hand: i. Using the MINIPREP: A. Check the spreader slide and clean if necessary. B. Place a clean slide on MINIPREP. C. Using a Diff safe or plain HCT tube, place a small drop of EDTA whole blood on the black spot. D. Push down on the handle to smear out blood. ii. By hand: A. Place a drop of EDTA whole blood on a clean slide. Page 3 of 13

4 B. Place a clean pusher slide at an angle right behind the drop and pull the angled slide into the drop of blood. C. Let the blood spread a little on the edge of the pusher slide and then quickly push the pusher slide toward the other end of the stationary slide. c. Manually label slide with patient name and sample order number, or place patient s ID aliquot sticker on slide assuring that the label does not extend over the edges of the slide. d. Place slide face up to dry the smear, or place face up on a slide warmer if available. Overall evaluation of slide: 1. After staining, examine the slide on a low power, X for quality. a. Check the overall staining of the slide for proper staining of red blood cells and inner structures of white blood cells. b. Estimate the WBC. If using a 40X or high dry objective, see attachment Lab for instructions. If using a 50X oil objective, see WBC Estimate below for instructions. c. Check the distribution of the WBCs. Is there an increase or decrease in any cell type? Are the WBCs at the feathered edge and edges representative of the rest of the slide? Larger cells and clumps of cells tend to be carried to the feathered edge. Among the cells likely to be found in this region are immature leukocytes, nucleated red cells, megakaryocyte nuclei, platelet clumps, mitotic forms, tumor cells, macrophages, microfilariae and monocytes and neutrophils containing red cells, bacteria and parasites. Are the abnormal cells found in the body of the slide? d. Are there any rare immature or abnormal nucleated cells? e. Are there platelet clumps, giant platelets or megakaryocyte fragments? If there are megakaryocyte fragments, the WBC count needs to be corrected if >5/100 WBC. See policy Lab-5130 Verification of XN9000/XS1000i Results, for instructions and calculations to correct the WBC. f. Look for platelet satellitism and agranular platelets. g. Check the RBC distribution for rouleaux and agglutination. h. Look for RBC features of: micro, macro, hypo, poly, poik, target cells and nucleated RBCs and RBC inclusions. When there are >5 NRBC/100 WBC, the WBC needs to be corrected if your analyzer does not correct for NRBCs or if the Manual NRBC count does not agree with the Automated. i. Look for a good area to do the differential. Scan: 1. Select an area just behind the feathered edge where the red cells are barely touching. 2. Scan the slide on 50X or 100X for seconds. 3. The Comment Electronic diff. verified by slide review can be resulted out with the absolute counts from the automated differential in lieu of a manual differential if the following criteria are met. a. For XN-9000 users the scan agrees comment can be used provided that the scan agrees with the electronic differential, there is not a vote out, there are 2 Metas, and no myelos, pros, or blasts. Page 4 of 13

5 b. For XS users the scan agrees comment can be used provided that the scan agrees with the electronic differential, there are not vote-outs, and there are no immature cells. c. For KX and Sysmex poch-100i users the scan agrees comment can be used provided that the scan agrees with the electronic differential, there are no vote-outs, there are no immature cells, and all results for Basos, Eos, and Monos are within normal limits. All automated results with a mixed cell result >11% should be scanned at a microscope. If the manual result values for Basos, Eos, or Monos are abnormal, report the manual differential. 4. Report the absolute values from the manual differential if: a. Presence of any immature cells, (sites using an XN instrument with an automated immature granulocyte count can use the automated results so long as the manual differential has 2 Metas) b. Vote out of electronic differential (partial or all) c. Manual differential varies from the electronic differential by greater than the 95% confidence limits as listed in attachment Lab d. Criteria met for two tech differential. NOTE for SYSMEX KX-21N and Sysmex poch-100i users: If mixed cell result >11%, perform scan. If results of scan show an abnormal amount of either Baso, Eos or Monos perform a manual differential. If the manual differential shows that all Baso, Eos and Monos are within normal limits, report electronic differential. If the Baso, Eos or Mono numbers are abnormal, report the manual differential. Counting Differential: 1. With the 50X oil or 100X oil objective, perform a differential by counting the first 100 WBCs located in sequence, using the manual counter, keyboard counter, or DI. Results are expressed in percent on the keyboard/counter. Absolute values are calculated by the LIS or by DI. 2. Criteria for Performing a 2 Tech Manual Differential - A second tech will perform another differential on slides with the following abnormal parameters. For regional/partner labs - If the two techs do not agree, remake the slides and repeat. If the two techs still do not agree, the sample and slides must be sent to LaCrosse for verification or reviewed by technical leader on-site. For La Crosse lab if the two techs do not agree, follow the instructions listed below for performing an RVIEW. a. Sysmex XN9000 users: i. Manual differential varies from electronic differential by more than the 95% confidence limits. See attachment Lab-? for electronic versus manual differential confidence limits chart. ii. NRBC % from XN and manual diff do not agree within 95% confidence limits if NRBC% is 40; or within 25% if NRBC % is > 40. See attachment Lab for chart. b. Sysmex XS-1000i Users i. Greater than 20% monos ii. Greater than 20% eos iii. Greater than 2% basophils Page 5 of 13

6 iv. Any immature cells or NRBC v. Any new adult or child > than 2 yrs. with > 50% lymphs vi. Greater than occ atypical lymph (occ = >5 30 per 100 WBCs) iii. Manual differential varies from auto differential by more than the 95% confidence limits. See attachment Lab for chart. c. Sysmex KX-21N and Sysmex poch-100i Users i. Greater than 15% monos ii. Greater than 10% eos iii. Greater than 2% basophils iv. Any immature cells or NRBC v. Any new adult or child > than 2 yrs. with > 50% lymphs vi. Greater than occ atypical lymph (occ = >5 30 per 100 WBCs) vii. Manual differential varies from auto differential by more than the 95% confidence limits. See attachment Lab for chart. 3. Criteria for review by technical leader, designated tech or medical director in LaCrosse. a. Sysmex KX-21N and Sysmex poch-100i Users i. Exceptionally abnormal RBC morphology with greater than 2+ morphology. ii. Immature cells more than 2% metas, eos or basos younger than band form; any blasts, immature lymphs or monos iii. Moderate or many atypical lymphs iv. Severe shift to the left greater than 30% bands (Bands and segmented neutrophils will be classed together as mature neutrophils. Severe Left shifts should still be verified by the technical leader, designated tech, or medical director in La Crosse.) v. Greater than 20% monos vi. Any smear with 5 or more NRBC vii. Any slide on which 2 techs do not agree within 95% confidence limits. See attachment Lab for chart. b. Sysmex XS-1000i Users i. Exceptionally abnormal RBC morphology with greater than 2+ morphology. ii. Immature cells more than 2% metas, eos or basos younger than band form; any blasts, immature lymphs or monos iii. Greater than 30% monos iv. Any smear with 5 or more NRBC v. Any slide on which 2 techs do not agree within 95% confidence limits. See attachment Lab for chart 4. Sysmex XN-9000 users - Criteria for performing an RVIEW: When the following criteria are met, a slide will be reviewed by the technical leader, designated tech (RVIEW tech), medical director, or hematopathologist. RVIEW techs have received additional training and completed additional competency testing to verify abnormal differentials in the absence of the technical leader or medical director. They will be a resource for students and new employees. a. Blasts (New finding within 1 year). b. If the "Two Tech Diff" doesn't agree. c. If unsure of cell ID or if referee is needed, consult with the hematopathologist of the day (daily or next AM) or the Inservice Hematologist (weekends/new diagnostic case). Page 6 of 13

7 d. Confirmation of blood parasites. e. Do NOT free text a description without input from the hematopathologist or Hematologist. 5. Types of Cells a. Segmented Neutrophil-(mature neutrophil). Medium size (10-15 um diameter) with two to five unequal nuclear lobes connected by a filament. Purple chromatin coarsely clumped with a few open areas randomly distributed. Pinkish tan or light lilac cytoplasmmay contain a few red-purple residual primary granules. b. Neutrophil band (counted as mature neutrophil). Similar to segmented neutrophil except for nuclear shape. The connection between the end of the beginning lobe formation of the nucleus is band-like rather than a filament. The connecting band is wide enough to contain nuclear material. If no nuclear material can be seen in the connecting band, the cell is a segmented neutrophil. c. Eosinophil: Similar to the segmented or band neutrophil except for a two-lobed nucleus and presence of numerous bright orange spherical cytoplasmic granules. Usually um in size. d. Basophil: Similar to the mature neutrophil except for a round or indented light red-purple staining nucleus and presence of large purple-black cytoplasmic granules that may obscure the nucleus um in size. e. Lymphocytes: Small to large (8-14 um) in size and mononuclear with a high nuclear cytoplasmic (N/C) ratio. Nucleus: Round, oval or slightly indented with semi-densely blocked red-purple chromatin, the larger the cell, the less dense in the nucleus, nucleolus occasionally visible, especially in larger cells and in thin areas. Cytoplasm: pale or medium blue, translucent, and may contain several azurophilic (red-purple) granules, larger lymphocytes have a smaller N/C ratio than small ones. Variant forms including reactive (dark cytoplasm or edges), immature (contains a nucleoli), indented/clefted nucleus, plasmacytoid lymphs and lymphs with cytoplasmic projections will be categorized at Atypical. f. Plasma cell: um in size with a round or oval shape and low N/C ratio. Nucleus is usually eccentrically located with semi-coarsely blocked chromatin similar to lymphocyte. The cytoplasm is medium to deep blue with a prominent perinuclear clear area. g. Monocyte: Medium to large (10-20 um) mononuclear, with a medium N/C ratio. Nucleus is usually convoluted or horseshoe-shaped, can be round, oval, indented, deeply lobulated, or even segmented. It contains evenly distributed chromatin that is pale and delicate staining. Cytoplasm is usually abundant, dull gray-blue with a few coarse redpurple or tiny dust-like granules may be present, causing a ground glass like appearance. Vacuoles are common and phagagocytized particles are sometimes seen. 6. NRBC and megakaryocytes: a. Nucleated red blood cells and megakaryocytes are counted during the 100 WBC differential. The DI keypad is designed to keep them separate from the 100 WBCs. When using a manual cell counter, keep them separate from the 100 WBCs. If NRBCs or megakaryocyte fragments are present, they are reported along with the manual differential. When there are more than 5 NRBCs or 5 Megakaryocytes or Megakaryocyte fragments per 100 WBCs, the total WBC count must be corrected using this formula: WBC times 100 divided by the number of NRBC or Megakaryocytes plus Page 7 of 13

8 100. A WBC corrected for the presence of NRBCs or megakaryocytes or fragments will be lower than the uncorrected value. **Example: The WBC count from the analyzer is 20,000. Multiply the WBC count by 100. (=2,000,000). There are 5 NRBC per 100 WBC and 10 Megakaryocyte fragments per 100 WBC found upon slide review. Add the number of mega frags and NRBC together and add 100 (=115). Divide 2,000,000 by 115 = corrected WBC of 17,391. Corrected WBC = WBC x 100 = 20,000 x 100 = 2,000,000 = 17,391 NRBC + Mega **If the WBC is corrected, absolute differential values must be calculated using the corrected WBC. b. Sysmex XN9000 users: i. Correlate the XN electronic %NRBC to the NRBCs seen in the manual diff. They must agree within the 95% confidence limit if the NRBC% is 40; or within 25% if the NRBC count is >40. ii. If counts do not agree within the stated limits, refer to NRBC section in Lab-5130 Verification of XN9000/XS1000i Results or attachment Lab to correct WBC and absolute counts. iii. If NRBCs or megakaryocyte fragments are present, they are reported along with the manual differential using the diff keypad. 7. Atypical Lymphocytes: a. Variant lymph forms including reactive (dark cytoplasm or edges), immature (contains a nucleoli), indented/clefted nucleus, plasmacytoid lymphs and lymphs with cytoplasmic projections will be categorized at Atypical. b. Up to 5% atypical lymphocytes are considered normal and do not need to be commented on. (5% of the total WBCs, not 5% of the total Lymphs.) Quantitate the number of atypical lymphs with descriptive terms: i. Occasional (occ) = >5 to 30% ii. Moderate (mod) = >30 to 60% iii. Many = >60% 8. Smudge and Basket cells: Smudge cells will be counted as WBCs in the manual diff. a. If the cell has a dense purple appearance, count as a lymph. If characteristics of a neutrophil, eos, or baso, count respectively as those cell types. b. If the manual diff agrees with the electronic diff report Electronic diff verified by slide review. and result smudge cells as "present". c. If the electronic diff is voted out, report the manual diff with smudge cells counted. Result smudge cells as "present". The ANC will have to be manually calculated if a Hemogram is being reported. d. An albumin prep diluting 4 drops of blood with 1 drop of 2% albumin can be made in rare cases when smudge cells are difficult to assess. If an albumin prep is needed, the sample should be sent to the La Crosse lab. This tool should not be routine practice because turnaround times are greatly affected. Page 8 of 13

9 i. Note: Albumin laced smears very easily flake off of the glass slides. Allow slides to dry completely before staining and examine them with great care. Wiping should be avoided. 9. WBC Estimate: a. Perform WBC estimation to confirm automated WBC. Note abnormal populations. b. To estimate WBC: i. Perform in an area where RBCs are just starting to overlap. ii. Average the number of WBCs from fields. iii. If using a 50X oil objective, multiply the average by 3. iv. If using a 40X or high dry objective, see attachment Lab for instructions. Red Cell Morphology: RBC morphology characteristics are measured and reported by scanning 10 fields in different areas of the slide with evenly dispersed red cells using the 100X oil immersion objective. This area is usually where cells are just touching or beginning to overlap. The test code RBC Morphology is reported as Normal or Present. The individual components of morphology are reported by using the following grading steps. Variance of one grading step is acceptable tech to tech variation when grading RBC morphology. 1. Uniform Grading System for common RBC abnormalities based on scanning at least 10, 100X oil immersion fields: Normal = 0-4 cells/100x field 1+ = 5-14 cells/100x field, slight but significant increase from normal 2+ = cells/100x field, moderate increase 3+ = greater than 30 cells/100x field, marked increase Morphology included in this grading range: Micro, Macro, Echino, Ovalo, Ellipto, and Stomatocytes. 2. Exceptions for the more uncommon RBC abnormalities: 1+ = 0-2 cells/100x field 2+ = 3-5 cells/100x field 3+ = greater than 5 cells/100x field Morphology included in this grading range: Hypo, Poly, Acantho, Schisto, Sickle, Sphero, Teardrop and Target cells. 3. Anisocytosis - Variation in size of RBC -anisocytosis is not reported; the specific cell types of microcytes and macrocytes will be quantitated as 1+, 2+ or 3+ using the grading guidelines listed above. Normal RBC size = 7 u +/- 1 µm Microcyte = < 6.0 µm Macrocyte = > 8.0 µm a. Macrocytosis is graded based on the number of cells/field that are greater than 8.0 µm. Microcytosis is graded based on the number of cells/field that are less than 6.0 µm. b. If you have trouble approximating the size of the RBC, use the micrometer eyepiece. c. After you have committed yourself to the degree of micro and/or macro, check the MCV for correlation. 4. Poikilocytosis- Variation in shape of the RBC poikilocytosis is not reported; the specific cell types will be quantitated as 1+, 2+ or 3+ using the grading guidelines listed above. Page 9 of 13

10 5. Polychromatophilia- Significant amount of immature red cells a. Polychromatic cells display various shades of gray-blue staining red cells indicating presence of basophilic ribonucleoprotein material b. Grade the number of cells, not the degree of basophilia, since this varies with technique used in staining. 1+ = 0-2 cells/100x field 2+ = 3-5 cells/100x field 3+ = greater than 5 cells/100x field 6. Hypochromia- Decrease in hgb content of RBCs a. Hypochromic cells are abnormally pale in color, that is, have an increased central pallor. b. When grading, consider both the degree of pallor and the number of cells involved. c. After you are committed to the degree of hypochromia, check the MCH and MCHC. An MCHC less than 30.0 should have hypochromic cells present. If there is a discrepancy, get another opinion or leave the smear for further investigation. Be careful about cells with artifacts, which would have sharp central borders. d. Use the same grading criteria as polychromatophilia. 7. Other Cellular Abnormalities - The following are not quantitated: a. WBC anomolies resulted as Present: Smudge Cells, Dohle Bodies, Toxic Granulation, Auer Rods, Vacuolated, Hypersegmented, Bilobed, and Agranular Neutrophils. b. RBC anomolies resulted as Present: Rouleaux, Basophilic Stippling, Pappenheimer Bodies, Howell-Jolly Bodies, and Cabot Rings. c. PLT morphology: resulted as Normal, Giant, and/or Agranular. 8. WBC/RBC Parasites or Bacteria a. Anaplasmosis is a tick borne parasite endemic in the US. Characteristic morulae or cytoplasmic inclusions are seen in the granulocytes. Ehrlichiosis is a tick borne parasite endemic in the US. Characteristic morulae or cytoplasmic inclusions are seen in the monocytes. b. Malaria: Various forms including rings with Schuffner's dot may be seen in the RBCs depending on the stage of the parasite's life cycle. c. Babesia: Multiple ring forms may be found in the RBC. Babesia lacks the brownish pigments in the rings, Schuffner's dot and multiple stages seen in malaria. d. Bacteria can be seen intra- or extra-cellularly. e. If any of the above are seen, they should be reported by resulting with a coded entry as follows: To add one of these comments in DI to a CBC report, select the patient and: i. Select Verify Run with cell counter. ii. Perform a manual differential cell count if indicated. iii. Disable the Cell Counter Keys iv. Add appropriate morphology and comments. v. Select the drop down box for the parameter Comment. vi. Choose See Note vii. Choose Send data thru system. viii. Go to the appropriate run for the Morphology Group. ix. Place the cursor on the test comment field for the See Note result. x. Choose the appropriate comment xi. Release results from the designated test groups to send results to the LIS Page 10 of 13

11 xii. If resulting in LIS, result test code COMMENT as See Note, then add a comment using the.beakh picklist in the comment tab. f. LaCrosse Lab - Refer to Lab-5190 Blood Parasites Malaria and Microfilaria or for special preparation and staining of slides for detection and confirmation. Platelet Estimation and Morphology: 1. Estimate whether platelet number appears normal, increased, or decreased and compare to electronic platelet count. 2. Platelet morphology is reported with every differential as Normal, Giant, and/or Agranular. a. Platelets are 2-3 µm in diameter with no nucleus and red-purple granules. b. Look for agranular platelets and giant platelets (platelets >6-8 µm). 3. Platelet estimate: a. Using 100X oil, find an area where the RBCs touch or just overlap. b. Count 10 fields and calculate the average. c. Multiply the average number of platelets per field by a "Factor". It takes practice to find a factor that works for you. i. Example: 10, 9, 10, 8, 5, 4, 9, 11, 9, 7 counted for a total of 82. Divide 82 by number of fields (10) = 8.2 average. ii. 8.2 multiplied by a factor of 12 = 98.4 for an estimated platelet count. 4. If platelet clumps are detected and the platelet count has been verified: a. Delete the platelet result in LIS. b. Enter the appropriate canned comment regarding platelet clumping and adequacy. c. Call the provider to report the change in result. d. Complete an event form and inform the technical leader. Regional/Partner samples needing verification: 1. Each regional/partner site has criteria listed previously in this policy for differential slide review by GHS-La Crosse laboratory. 2. The regional/partner sample should be accompanied by a completed Regional/Partner Result Verification Form (Lab Differential Counting and Morphology), the regional/partner hematology analyzer printout, the EDTA tube of blood and slides, stained and unstained. 3. See the Regional/Partner Result Verification Form (Lab ) for complete handling instructions. Electronic Differential to Manual Differential Comparison 1. The Sysmex Hematology instruments that provide an electronic differential are checked against a tech performed 100 cell manual differential twice a year for correlation of results. This can be performed by lab staff following the Instrument specific procedures in the policies listed below. a. Lab-1535 Complete Blood Count of Whole Blood on the Sysmex KX-21N b. Lab-1501 Sysmex XS-1000i Procedure c. Lab-5135 Sysmex XN-10 Complete Blood Count and Parameters - Whole Blood PROCEDURE NOTES: Variance of one grading step is acceptable tech to tech variation when grading RBC morphology. Page 11 of 13

12 CALCULATIONS: Manual ANC = WBC x (%Seg + %Band) XN-9000 users: ABIG = WBC x (%Meta + %Myelo + %Pro) If additional calculations are needed refer to policy Lab-5130 Verification of XN9000/XS1000i Results. INTERPRETATION: 1. Expected normal values for differentials: Mature Immature LYMPHS MONOS EOS BASOS Neutrophils Grans Age Group K/uL K/uL K/uL K/uL K/uL K/uL up to: Hour Month Month Year Year Year Year Year Year Year Year Year Adult Total IG Myelos Metas Pros Blasts Age Group up to: K/uL K/uL K/uL K/uL K/uL 48 Hours Days >30 Days Page 12 of 13

13 2. Expected normal range for platelets is K/uL. REPORTING RESULTS: 1. Verify that your results and estimations agree with electronic results, including: a. MCHC/hypochromia b. MCV/anisocytosis/macro/micro c. WBC/estimate d. Plt/estimate e. HGB/polychromasia, etc. 2. LIS or DI - After finishing slide review, the CBC is completed by resulting a Manual differential or Electronic diff verified by slide review. RBC and platelet morphology are also resulted. The Complete comment should be verified last and will remove the specimen from the worklist. LIMITATIONS: N/A REVIEW AND CHANGES: This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical Director or designated person. Changes require retyping document or form and review by the Medical Director. REFERENCES: 1. Todd & Sanford, Clinical Diagnosis by Laboratory Methods, 15th Edition. 2. Hematology, Basic principles and practice, Second Edition. 3. CAP Guidelines for Evaluation. 4. Clinical Laboratory Technical Procedure Manuals, NCCLS, 2nd Ed. Approved guidelines, July 1992, Vol. 12, NO Reference Leukocyte Differential Count (Proportional) and Evaluation of Instrumental Methods, approved standard, NCCLS, March 1992, H20-A, Vol.12, NO A Color Atlas and Instruction Manuel of Peripheral Blood Cell Morphology, Barbara O Connor, Williams & Wilkins, Procedure for 50X WBC Estimation was obtained from Children s Hospital, Milwaukee, WI. 8. Electronic Versus Manual Differential Chart; obtained from Western Wisconsin Institute, MLT Program materials; 10/ Nathan & Oski s, Hematology of Infancy and Childhood, 7 th Edition. p Appendix 11, p Appendix 26. Page 13 of 13