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AKHK SOP NO.: EMT 903 Version: 00 Prepared by: Patience Kache Date: Reviewed by: Jacob Seroni Date: Effective Date:
CRITICAL VALUE REPORTING- AKHK Kenya
Verified by: Milton Isanya Date: Approval authority: Dr. B. V. S. Prasad Date: Next Review Date:
DOCUMENT CHANGE RECORD Date Rev. Section Description/ Reason for Change
PURPOSE AND APPLICABILITY 1.1.0. To outline the procedure of performing differential count on a PBF SUMMARY Examination of the well-prepared and well-stained blood smear is one of the most efficient and important diagnostic screening tests used in the evaluation and detection of abnormal blood counts. Equally important is the blood morphology, which can often reveal important diagnostic or therapy-related information. Therefore, the manual differential white blood cell count is performed to determine the relative number of each type of white blood cell present in the blood. At the same time, a study of red blood cell, white blood cell, and platelet morphology and maturity is performed. This includes an approximation of the platelets count. This is a CONTROLLED document. Any SOP not stamped in blue is not in use. Page 1 of 14
Cell differential counter 4.2.5. Monitor uniformity standards through scheduled competency assessment. Wright stained peripheral smear with identification label (attached to the smear side only) 4. if there are an increased number of leukocytes in this area. in the center and results in inaccurate manual differential counts. 6. Page 2 of 14 . such as lymphocytes. 5. However. The following steps must be taken during differential testing to ensure uniform reporting within the group: Agree upon the definition of all terms used.0. RESPONSIBILITIES PROCEDURE 6.0. Establish criteria for peripheral smear review by the department supervisor and pathologist 6. another blood smear must be prepared and evaluated. A proper wedge peripheral blood smears stained in accordance with the Wright Stain (a Romanowsky type blood stain) manual or automated methods.0.0.3.4.0. Verify that the automated CBC report and blood smear identification match. This is a CONTROLLED document. the differential count may be inaccurate.2.6. Immersion Oil 3.0. Examine the feathered edge (figure 1) of the smear to determine blood film adequacy.1.1.1. Place the stained slide (specimen side up) on the microscope stage.0. The 10X low power objective.0. Evaluate the number of white cells per 100X at the feathered edge. Circulate unfamiliar slides among the group members and compare results. Quality Control: 6. Microscope Low power 10X objective and 100X oil immersion objectives 4.0. The edges of even the best-prepared blood smear have accumulations of leukocytes. labeled automated CBC instrument report with histograms 4. 18.104.22.168. 4.DEFINATIONS AND ABREVIATIONS EQUIPMENT/TOOLS/SPECIMEN 4. Incorrectly prepared films may have too many large cells at the film edges. Reporting: 6. 4.2. leaving relatively smaller cells.1.0.0. Establish a required level of accuracy for new laboratory personnel and assess competency before performing patient differential count testing. if it exceeds 2-3 times the number per field in the body of the film. Any SOP not stamped in blue is not in use.2.
2. The field will also be devoid of broken areas caused by improper smear preparation. Do not perform a platelet estimate. Select the optimal counting area. MANUAL DIFFERENTIAL: 6.2.3. 6. Review the area where the red cells are evenly distributed and begin to overlap.0. Review all abnormally large leukocytes under 100X for appropriate classification and follow up testing. 6. Scan for abnormalities such as platelet clumping.1. This is the area where the erythrocytes are adjacent but do not overlap. The erythrocytes will be evenly distributed and not distorted. the automated platelet count will be falsely decreased. refer to platelate clumping in this SOP for the appropriate corrective action. rouleaux or agglutination is identified.If platelet clumps are identified in this area. 6. Use a differential cell counter. agglutination or abnormally large leukocytes.4. If platelet clumping. Follow the procedure for platelet clumping as identified later. rouleaux.2. 22.214.171.124. Any SOP not stamped in blue is not in use. Perform the 100 cell differential count. Page 3 of 14 . The 40x objected may be used to perform the differential count but the 100x objective must be utilized to classify any immature cells to include myelocytic cells more immature than the segmented neutrophil. count and classify 100 white blood cells using the cross-sectional technique where the white cells are counted in consecutive fields as the blood film is moved from side to side as pictured This is a CONTROLLED document. Select the best area for detailed morphological evaluation and differential count.
e. a 50 cell count can be performed and converted to 100% by multiplying all values by 2. or monocyte. Multipy the decimal by the total white count. i.. myelocyte. stain and recount the differential on the new slide. Any SOP not stamped in blue is not in use. enumerate them separately from the white blood cell count. do not certify the manual differential until it can be verified by the pathologist. Note: If blasts or promyelocytes are identified on the peripheral smear. metamyelocyte. Round accordingly and report only whole numbers.e. If nucleated red blood cells (NRBC) or Megakaryocytes are seen during the differential count. Convert the total percentage to decimal. i. If the manual differential count does not correlate to within 10% (10 cell difference) of the automated differential count for any one cell class. Classify the cells as follows: segmented neutrophil. review the slide for accurate wedge smear preparation. proceed as follows: Enter the manual differential count on the Patient report.45 Lymphs = 4. If the automated WBC count is greater than 30. This is a CONTROLLED document. repeat the count. a 200 cell count must be performed and converted to 100% by dividing all values by 2.0 K/uL WBC x 0.4.e. Add the following comment on the report 200 cells counted during the manual differential and converted to percentage.0 K/uL.0 K/uL.3.3. eosinophil. 6. 10. If the slide is acceptable. Lymphocytes + Atypical Lymphocytes = Total number of Lymphocytes. 45% lymphs = 0.6. Neutrophils + Bands + Meta¶s. If the slide quality is questionable. etc = Total number of Neutrophils. Calculate absolute cell counts using the manual differential. Add each cell class to obtain a total number for that class. i. prepare. Add the following comment on the report50 cells counted during the manual differential and converted to percentage. Page 4 of 14 . banded neutrophil.5 K/uL Lymphocytes. If the automated WBC count is less than 2. lymphocyte.45. basophil.3. If the recount is out .
Review a minimum of 10 fields. 6. do not amend the results.Add the totals from each cell class calculation and they will equal the total WBC count.0. Enter the manually calculated absolute counts from step above.4. in place of the automated absolute count fields on patient result with the following comments: Absolute counts calculated based on manual differential. Report Normocytic if microcytosis and/or macrocytosis are not present or they are present at less than 10 cells per field. the calculations were performed incorrectly and must be repeated. Locate a mature (normal and not atypical) lymphocyte for comparative purposes and review the smear for the following: 6. Result the manual differential 6.4.larger than normal 6. Begin the examination in an area where the red cells are touching but not overlapping. Page 5 of 14 . 6.3.9 3+ Many >21 RDW This is a CONTROLLED document.5. The test will be performed using the high power (100X) oil immersion objective.0 to 20.Variation in cell size is measured using the Red Cell Distribution Width (RDW) and is graded during the RBC Morphology Examination. and compare the RBC size to that of a normal lymphocyte. Tally the number of each classification per field and report as follows: 1+ 2+ 3+ Occasional 5-25% Moderate 25-50% Many >50% 10 to 50 cells 51 to 100 cells >100 cells Normocytic cells are not graded.normal size Microcytic . RBC Morphology and WBC Review The RBC Morphology and WBC Review will be performed with every manual differential. Red Blood Cell Size ± Normal erythrocytes are 6 to 8 micron in diameter or approximately the same size as the nucleus of a normal lymphocyte.5 If the CBC was certified.0 to 17.9 2+ Moderate 18. using the 100X oil immersion objective.2. Anisocytosis . Any SOP not stamped in blue is not in use. Grade the variation using the guide listed below: 1+ Occasional 15. There are approximately 300 RBC¶s present in this area in patients with a normal hematocrit. 6.4.4. The cells are classified and reported as follows: Normocytic .1.smaller than normal Macrocytic .3.3. If not.
Poikilocytosis is the term used to describe variation in cell shape.6. Tally the number of each classification per field and report as follows: Hypochromia 2+ Moderate 25-50% 51 to 100 cells Polychromasia 2+ Moderate 1 ± 2.Crenated or Burr Cells Elliptocytes or Ovalocytes Envelope forms or folded cells Schistocytes Spherocytes Stomatocytes Review the RBC shape in a minimum of 10 fields using the 100X oil immersion objective. Grade poikilocytosis using the following cell shape classifications identified below.Normal RBC¶s are red to buff pink with more color intensity at the marginal areas than in the central portion when stained with Wrights Stain.pale color and large central pallor Polychromasia ± bluish color and usually macrocytic Review the RBC color in a minimum of 10 fields using the 100X oil immersion objective.normal color Hypochromic . Cells are classified as follows: Normochromic . Page 6 of 14 . Report Normochromic if Hypochromia is not present or it is present at less than 10 cells per field. Red Blood Cell Shape ± Normal RBC¶s are round with an area of central pallor that occupies one-third of the cell. Polychromasia can be reported with both normochromic and Hypochromic reports.5% 2 to 5 cells 1+ Occasional 5-25% 10 to 50 cells 3+ Many >50% >100 cells 1+ Occasional 1% <1 to 2 cells 3+ Many >2. Any SOP not stamped in blue is not in use.4.4.5. Tally the number of each classification per field and report as follows: This is a CONTROLLED document. Red Blood Cell Color .4. Acanthocytes Bite Cells Blister Cells Codocytes ± Target Cells Dacryocytes ± Teardrops Drepanocytes ± Sickle Cells Echinocytes . 6.5% >5 cells Normochromic cells are not graded.
Tally the number of each classification per field and report as follows: RBC Inclusions 2+ Moderate 1 ± 2. Bite Cells. and the associated clinical conditions. Envelope forms or Folded Cells.Codocytes (Target Cells).5% >5 cells This is a CONTROLLED document. Red Blood Cell Inclusions ± Various RBC inclusions can occur for a variety of reasons. grade and report poikilocytosis as 1+ occasional. Blister Cells.5% 2 to 5 cells 1+ Occasional 1% <1 to 2 cells 3+ Many >2. Echinocytes (Burr or Crenated).6. Drepaocytes (Sickle Cells). Tally the number of each inclusion classification per field and report as follows: Basophilic Stippling Howell Jolly Bodies Pappenheimer Bodies Cabot Rings Hemoglobin C Crystals Bacteria Parasites Mauer¶s Dots Schüffner¶s Dots Nucleus ± Refer to paragraph for procedure.5% Many >2. & Spherocytes 1+ 2+ 3+ Occasional 1% Moderate 1 ± 2. Elliptocytes (Ovalocytes) & Stomatocytes 1+ 2+ 3+ Occasional 5-25% Moderate 25-50% Many >50% 10 to 50 cells 51 to 100 cells >100 cells Acanthocytes. Page 7 of 14 . Artifacts Review the RBC shape in a minimum of 10 fields using the 100X oil immersion objective. 6. Dacryocytes (Teardrops). Any SOP not stamped in blue is not in use.4. using Wright Giemsa Stain. Scan a minimum of 10 fields using the 100X oil immersion objective. 2+ Moderate. Refer to appendix 3 for a brief summary of the various inclusion appearances. 3+ Many.5% <1 to 2 cells 2 to 5 cells >5 cells If none of the shapes identified above are present or they are present at less than the minimum criteria. Schistocytes.
If any of these inclusions are present.4. Similar to mature neutrophil. Numerous bright orange spherical granules Large purple-black granules that may obscure the nucleus This is a CONTROLLED document. Mauer¶s Dots. Similar to mature neutrophil except the nucleus is bi-lobed. may contain few red-purple primary granules. Same as above but may be more basophilic with more primary granules.7. specific non-visible neutrophil granules.Bacteria. Notify the pathologist immediately to arrange verification and provider notification. parasite. Similar to mature neutrophil except nuclear shape is a nonsegmented continuous band of chromatin that may contain more open areas. Page 8 of 14 . 6. CYTOPLASMIC COLOR Pinkish tan to light lilac. Purple chromatin coarsely clumped with randomly distributed open areas. THEY REQUIRE IMMEDIATE PATHOLOGIST CONFIRMATION before they can be reported. Any SOP not stamped in blue is not in use. TEXTURE. and Schuffner¶s Dots are critical findings. White Blood Cell Color CELL TYPE & SIZE Segmented Neutrophil 10-15 Qm Banded Neutrophil 10-18 Qm Eosinophil 10-15 Qm Basophil 10-15 Qm NUCLEUS COLOR. & SHAPE Two to five nuclear lobes connected by filaments.
Nucleus may be stretched like a band across the cell. increased number of azurophilic granules. Neutrophilic Inclusions ± Various neutrophil inclusions can occur for a variety of reasons. Tally the number of each inclusion classification per field and report as follows: Döhle Bodies Toxic Granulation Alder¶s Anomaly Chédiak-Higashi Anomaly Auer bodies Vacuoles Phagocytized material Review the Neutrophils in a minimum of 10 fields using the 100X oil immersion objective. Low N/C ratio. evenly distributed pale and delicate chromatin. Chromatin is coarse and clumped with dense masses arranged in a wheel-like pattern. usually convoluted or horseshoeshaped. Usually abundant. usually eccentric. may have vacuoles of varying size.4. Any SOP not stamped in blue is not in use. The nucleus may have visible nucleoli. dull gray.blue. Medium to deep blue and occasionally flame color with a prominent perinuclear clear area or hof. CYTOPLASMIC COLOR Lymphocyte 7-15 Qm Pale or medium blue. Mononuclear. and may contain several azurophilic granules Atypical Lymphocyte 10-25 Qm Abundant cytoplasm with vacuoles. Irregularly shaped or unusual chromatin pattern. may be binucleated. Scan a minimum of 10 fields using the 100X oil immersion objective. Nucleus is oval. vacuolated. A small pale chromocenter may be present. Round to ovoid.CELL TYPE & SIZE NUCLEUS COLOR. using Wright Giemsa Stain. TEXTURE. round. translucent. sparse lilac granules Plasma Cell 10-20 Qm Monocyte 12-20 Qm 6.8. radial and peripheral basophilia. and the hematopoietic cells involved. agranular. Chromatin is diffusely dense or coarse with clumpy masses. Tally the number of each classification per field and report as follows: This is a CONTROLLED document. or slightly indented. rarely multinucleated. & SHAPE Mononuclear with a high N/C ratio. Page 9 of 14 . Nucleoli are non-visible. Refer to appendix 4 for a brief summary of the various inclusion appearances and morphology.
5% 2 to 5 cells 3+ Many >2. Page 10 of 14 .5. For example.5.5. pince-nez or mononuclear) Hypersegmentation (6 lobes) Monocytoid change Monocytes Granulocytic change Hyperlobulation If any of the dsyplastic features are present. rouleaux formation can be reported as present. tally the number of each classification per field and report.2. Any SOP not stamped in blue is not in use. Severe hemolysis usually manifests with increased platelet counts that cannot be confirmed by the peripheral smear examination.4. 6. if 4 hypersegmented neutrophils are identified during the 100 cell differential count.4. This is a CONTROLLED document. Dysplastic Features± The following list of abnormalities can occur in the following cell types: Granulocytes: Agranular Abnormally large granules Hyposegmentation (bi-lobed. It can be observed in certain hemolytic anemias. prepare and evaluate a new blood smear. report ³4% Hypersegmented neutrophils present´ and include the 4 cells in the neutrophil count for the 100 cell differential cell count. 6. Rouleaux Formation ± This is observed in the normal examination area. Rouleaux occurs as a result of abnormal RBC arrangement due to biconcave surface opposition.0. If they are not appropriate corrective action must. 6. It is commonly seen in multiple myeloma and hyperproteinemia. The erythrocytes appear in short or long stacks resembling coins or flat plates.5. 6. Review the hemogram indicies for rule of three acceptability.1+ Occasional 1% <1 to 2 cells Neutrophilic Inclusions 2+ Moderate 1 ± 2. Provide the number and type of cells as a percentage of 100 cells.5% >5 cells 6.3. 6. Repeat for each dysplastic feature noted above. If there is considerate difference between the RBC indicies and blood smear presentation. Spurious result identification ± Engage the 40X oil immersion objective and review the slide for the any of the abnormalities identified below. Hemolysis ± Mild hemolysis is frequently undetected in most CBC tests. Reject hemolyzed samples and refer to the Spurious Results SOP for further guidance. If the indicies are within range.1. atypical pneumonia. staphylococcal infections and trypanosomiasis.9.. Agglutination ± This occurs as cells aggregate into random clusters or masses when exposed to various red cell antibodies.5.
2.1. Notify the Ward/OPD.6. Platelet Estimation 6. Perform a platelet estimate on any differential or RBC Morphology review smears. If this occurs follow the steps below: Values greater than 50 x 10/L: Document that "Platelet count inaccurate due to platelet clumping or platelet satellitism present". Do a manual platelet estimate count if a significant numbers of Microcytic red blood cells and/or small cell fragments are detected during the RBC morphology. This is due to thrombocytopenia or micro fibrin clotting. Occasionally platelets will be satellite around the neutrophils. If a significant numbers of giant platelets and/or platelet clumps are detected. clumps or platelet satellitism. Platelets Between 2-3 Qm in diameter Between 3-6 Qm in diameter Cytoplasmic fragments with red-purple granules and no nucleus Usually round. Page 11 of 14 .7.0.6. the instrument count. Follow the above step until 10 fields have been counted. This is done to correlate the automated platelet count with the estimate and check the smear for giant platelets. and the platelet estimate process will be repeated.0. Count all the platelets in that area. WBC Correction for NRBC or Megakaryocyte presence .6. If the platelet count from the instrument does not correlate with the platelet estimate on the smear. Any SOP not stamped in blue is not in use. Another specimens requested for evaluation".1.6. the smear. containing multiple red-purple granules Megakaryocytes 6. locate an area of approximately 150 red blood cells. giving a halo or a platelet clumping appearance. Clinic or Physician that the platelet count is inaccurate. 6.If 10 or more NRBCs or Megakaryocytes are present use the below formula: WBC count X 100 = Corrected WBC Count # NRBCs or Megakaryocytes + 100 This is a CONTROLLED document. Calculations: 6.7. Refer to Calculation Section of this SOP for example. then multiply by (x) 20. The cells should be just touching or barely overlapping. To do this. Divide the total number by 10 to obtain the average platelet per field. Request new EDTA and Citrate tubes for evaluation Document that "Platelet count inaccurate due to platelet clumping or platelet satellitism present. a peripheral estimate of WBCs should be done to prevent reporting spuriously high white blood cell count.000 to obtain the platelet estimate. Keep the total number of all 10 areas counted.
000 For example: Total of 10 field counted = 100 platelets Average platelet count = 100 / 10 = 10 Platelet estimate = 10 X 20. Smudge or Basket cells: Cells that are obviously white blood cells in origin.8.. follow the below steps: This estimate is done on 100X oil immersion where the red blood cells are just beginning to touch each other. Refer any unusual smear to the supervisor and/or the pathologist. This will markedly reduce smudge formation and aid in diagnosis. 6. An increased number of these cells are seen in lymphocytic leukemia.2 X 10/L Corrected WBC 10 + 100 Document that the WBC count was corrected due to NRBCs or Megakaryocytes present.e. This is a CONTROLLED document.7. Note: Pyknotic cells: The nuclear chromatin condenses into a solid. To perform platelet estimation. Outlying clinics are required to submit smears they have prepared from fresh specimens when requesting CBCs through this laboratory. structureless mass.0. To do this. Count the number of WBCs in ten (10) fields of a stained smear.example: 20 X 100 WBC count = 20 X 10/L NRBCs = 10 = 18. If necessary prepare a new blood film using a 1:5 ratio of 22% albumin to whole blood.1. They are usually found in old blood i. Any SOP not stamped in blue is not in use. Page 12 of 14 . Interpretation & Reporting Results: After the 100 cells count. Peripheral smears will be kept for seven (7) days to assure further . RBC morphology and platelet estimation is completed. from outlying clinics. THEY ARE NOT COUNTED IN THE DIFFERENTIAL. resulting in a loss of characteristic shapes and forms thereby precluding identification of the cells. Smudge or basket cells are reported as Present.000 = 200 X 10/L Peripheral estimation of the WBCs should be done to check the accuracy of the actual count. follow the below example: Platelet estimate = Average of platelets in 10 field counted X 20. Take the average of this count and use the following table: 6. reviews the results and report.
0.verifications of the smear if requested by the physician. Abnormals kept longer 6. March 1992. may be distributed in a nonrandom manner on a blood film. Lea and Febiger Book Publisher. 7. Limitation of the procedure: Although morphologic evaluation of a stained blood film is one of the most important hematologic procedures. Vol.0. Pages 223 to 238. Second Edition.10.0. Hematology: Basic Principles and Practice. uncontrollable errors in cell distribution on the blood film. Page 13 of 14 . REFERENCES: 7. NCCLS Document H20-A. Churchill Livingston Inc. Hoffman. 19126.96.36.199. High percentages of these cell types. Reference Leukocyte Differential Count. 1993. Ninth Edition. 8. 7.. 1993..3. Brown. Section/Area Quality Management Front office/Dispatch Specimen Management Hematology Section Clinical Chemistry Blood Bank Microbiology Histology & Cytology This is a CONTROLLED document.2.0. Ronald. Any SOP not stamped in blue is not in use. Windrobe¶s Clinical Hematology. 12 No. the manual differentiation of leukocytes is an inaccurate and un-reproducible method. Lee. 7.. issued Date Issued Sign.9. Richard G. 1.0. It is subject to errors that cannot be eliminated such as sampling errors. particularly eosinophils and Basophils. Sources of error: Certain cell types.0. 7. Pages 308 to 312. Sixth Edition. Barbara A. especially if found in a limited area of the film. Lea and Febiger Book Publisher. should be rechecked before the numbers are reported. 6.0. Pages 102 to 105. and human inconsistency in cell interpretation. DISTRIBUTION LIST No. Hematology: Principles and Procedures.
Page 14 of 14 .0.9. Any SOP not stamped in blue is not in use. TRAINING LOG Document read by Signature Date This is a CONTROLLED document.
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