DIFF COUNT SOP

THE AGA KHAN HOSPITAL KISUMU.

PATHOLOGY DEPARTMENT

AKHK SOP NO.: EMT 903

CRITICAL VALUE REPORTING- AKHK Kenya

Version: 00

Prepared by: Patience Kache

Date:

Verified by: Milton Isanya

Date:Reviewed by: Jacob SeroniDate:

Approval authority: Dr. B. V. S. PrasadDate:

Effective Date:Next Review Date:

DOCUMENT CHANGE RECORD

Date Rev. Section Description/ Reason for Change

1.0. PURPOSE AND APPLICABILITY1.1.0. To outline the procedure of performing differential count on a PBF

2.0. SUMMARYExamination 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. of 14

3.0. DEFINATIONS AND ABREVIATIONS4.0. EQUIPMENT/TOOLS/SPECIMEN

4.1.0. A proper wedge peripheral blood smears stained in accordance with the Wright Stain (a Romanowsky type blood stain) manual or automated methods.

4.2.0. MicroscopeLow power 10X objective and 100X oil immersion objectives

4.3.0. Wright stained peripheral smear with identification label (attached to the smear side only)

4.4.0. Cell differential counter 4.5.0. labeled automated CBC instrument report with histograms

4.6.0. Immersion Oil

5.0. RESPONSIBILITIES6.0. PROCEDURE

6.1.0. Quality Control: 6.1.1. The following steps must be taken during differential testing to ensure

uniform reporting within the group: Agree upon the definition of all terms used. Circulate unfamiliar slides among the group members and compare results. Establish a required level of accuracy for new laboratory personnel and assess competency before performing patient differential count testing. Monitor uniformity standards through scheduled competency assessment. Establish criteria for peripheral smear review by the department supervisor and pathologist

6.2.0. Reporting:6.2.1. Verify that the automated CBC report and blood smear identification

match.6.2.2. Place the stained slide (specimen side up) on the microscope stage.

The 10X low power objective. Examine the feathered edge (figure 1) of the smear to determine blood film adequacy.The edges of even the best-prepared blood smear have accumulations of leukocytes. However, if there are an increased number of leukocytes in this area, the differential count may be inaccurate. Incorrectly prepared films may have too many large cells at the film edges, leaving relatively smaller cells, such as lymphocytes, in the center and results in inaccurate manual differential counts. Evaluate the number of white cells per 100X at the feathered edge, if it exceeds 2-3 times the number per field in the body of the film, another blood smear must be prepared and evaluated.


If platelet clumps are identified in this area, the automated platelet count will be falsely decreased. Do not perform a platelet estimate. Follow the procedure for platelet clumping as identified later.

6.2.3. Review the area where the red cells are evenly distributed and begin to overlap. Scan for abnormalities such as platelet clumping, rouleaux, agglutination or abnormally large leukocytes. If platelet clumping, rouleaux or agglutination is identified, refer to platelate clumping in this SOP for the appropriate corrective action. Review all abnormally large leukocytes under 100X for appropriate classification and follow up testing.

6.2.4. Select the best area for detailed morphological evaluation and differential count. The erythrocytes will be evenly distributed and not distorted. The field will also be devoid of broken areas caused by improper smear preparation.

6.3.0. MANUAL DIFFERENTIAL: 6.3.1. Perform the 100 cell differential count.

Select the optimal counting area. This is the area where the erythrocytes are adjacent but do not overlap. 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.

6.3.2. Use a differential cell counter, 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


6.3.3. Classify the cells as follows: segmented neutrophil, banded neutrophil, metamyelocyte, myelocyte, eosinophil, basophil, lymphocyte, or monocyte.

Note:If blasts or promyelocytes are identified on the peripheral smear, do not certify the manual differential until it can be verified by the pathologist. If the automated WBC count is less than 2.0 K/uL, a 50 cell count can be performed and converted to 100% by multiplying all values by 2. Add the following comment on the report 50 cells counted during the manual differential and converted to percentage.If the automated WBC count is greater than 30.0 K/uL, a 200 cell count must be performed and converted to 100% by dividing all values by 2. Round accordingly and report only whole numbers. Add the following comment on the report 200 cells counted during the manual differential and converted to percentage.If nucleated red blood cells (NRBC) or Megakaryocytes are seen during the differential count, enumerate them separately from the white blood cell count.

6.3.4. If the manual differential count does not correlate to within 10% (10 cell

difference) of the automated differential count for any one cell class, review the slide for accurate wedge smear preparation. If the slide quality is questionable, prepare, stain and recount the differential on the new slide. If the slide is acceptable, repeat the count. If the recount is out , proceed as follows:Enter the manual differential count on the Patient report.Calculate absolute cell counts using the manual differential.Add each cell class to obtain a total number for that class, i.e. Lymphocytes + Atypical Lymphocytes = Total number of Lymphocytes, Neutrophils + Bands + Meta’s, etc = Total number of Neutrophils. Convert the total percentage to decimal, i.e. 45% lymphs = 0.45. Multipy the decimal by the total white count, i.e., 10.0 K/uL WBC x 0.45 Lymphs = 4.5 K/uL Lymphocytes.


Add the totals from each cell class calculation and they will equal the total WBC count. If not, the calculations were performed incorrectly and must be repeated.

6.3.5. Enter the manually calculated absolute counts from step above. in place of the automated absolute count fields on patient result with the following comments: Absolute counts calculated based on manual differential.

6.3.5 If the CBC was certified, do not amend the results. Result the manual differential

6.4.0. RBC Morphology and WBC ReviewThe RBC Morphology and WBC Review will be performed with every manual differential. The test will be performed using the high power (100X) oil immersion objective. Begin the examination in an area where the red cells are touching but not overlapping. There are approximately 300 RBC’s present in this area in patients with a normal hematocrit. Locate a mature (normal and not atypical) lymphocyte for comparative purposes and review the smear for the following:

6.4.1. 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. The cells are classified and reported as follows:Normocytic - normal sizeMicrocytic - smaller than normalMacrocytic - larger than normal

6.4.2. Review a minimum of 10 fields, using the 100X oil immersion objective, 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 cellsNormocytic cells are not graded. Report Normocytic if microcytosis and/or macrocytosis are not present or they are present at less than 10 cells per field.

6.4.3. Anisocytosis - Variation in cell size is measured using the Red Cell Distribution Width (RDW) and is graded during the RBC Morphology Examination. Grade the variation using the guide listed below:

1+ 2+ 3+Occasional Moderate Many

RDW 15.0 to 17.9 18.0 to 20.9 >21


6.4.4. Red Blood Cell Color - 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. Cells are classified as follows:Normochromic - normal colorHypochromic - 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. Tally the number of each classification per field and report as follows:

Hypochromia1+ 2+ 3+

Occasional 5-25% Moderate 25-50% Many >50%10 to 50 cells 51 to 100 cells >100 cells

Polychromasia1+ 2+ 3+

Occasional 1% Moderate 1 – 2.5% Many >2.5%<1 to 2 cells 2 to 5 cells >5 cells

Normochromic cells are not graded. Report Normochromic if Hypochromia is not present or it is present at less than 10 cells per field. Polychromasia can be reported with both normochromic and Hypochromic reports.

6.4.5. Red Blood Cell Shape – Normal RBC’s are round with an area of central pallor that occupies one-third of the cell. Poikilocytosis is the term used to describe variation in cell shape. Grade poikilocytosis using the following cell shape classifications identified below.AcanthocytesBite CellsBlister CellsCodocytes – Target CellsDacryocytes – TeardropsDrepanocytes – Sickle CellsEchinocytes - Crenated or Burr CellsElliptocytes or OvalocytesEnvelope forms or folded cellsSchistocytesSpherocytesStomatocytesReview the RBC shape in a minimum of 10 fields using the 100X oil immersion objective. Tally the number of each classification per field and report as follows:


Codocytes (Target Cells), Echinocytes (Burr or Crenated), 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, Bite Cells, Blister Cells, Dacryocytes (Teardrops), Drepaocytes (Sickle Cells), Envelope forms or Folded Cells,

Schistocytes, & Spherocytes1+ 2+ 3+


If none of the shapes identified above are present or they are present at less than the minimum criteria, grade and report poikilocytosis as 1+ occasional, 2+ Moderate, 3+ Many.

6.4.6. Red Blood Cell Inclusions – Various RBC inclusions can occur for a variety of reasons. Scan a minimum of 10 fields using the 100X oil immersion objective. Tally the number of each inclusion classification per field and report as follows:

Basophilic StipplingHowell Jolly BodiesPappenheimer BodiesCabot RingsHemoglobin C CrystalsBacteriaParasitesMauer’s DotsSchüffner’s DotsNucleus – Refer to paragraph for procedure.ArtifactsReview the RBC shape in a minimum of 10 fields using the 100X oil immersion objective. Refer to appendix 3 for a brief summary of the various inclusion appearances, using Wright Giemsa Stain, and the associated clinical conditions. Tally the number of each classification per field and report as follows:

RBC Inclusions1+ 2+ 3+



Bacteria, parasite, Mauer’s Dots, and Schuffner’s Dots are critical findings. If any of these inclusions are present, THEY REQUIRE IMMEDIATE PATHOLOGIST CONFIRMATION before they can be reported. Notify the pathologist immediately to arrange verification and provider notification.

6.4.7. White Blood Cell Color

CELL TYPE

& SIZE

NUCLEUS COLOR,TEXTURE, & SHAPE

CYTOPLASMIC COLOR

Segmented Neutrophil10-15 m

Two to five nuclear lobes connected by filaments.

Purple chromatin coarsely clumped with randomly distributed open areas.

Pinkish tan to light lilac; specific non-visible

neutrophil granules; may contain few red-purple

primary granules.

BandedNeutrophil10-18 m

Similar to mature neutrophil except nuclear shape is a non-segmented continuous band of

chromatin that may contain more open areas.

Same as above but may be more basophilic with more

primary granules.

Eosinophil10-15 m

Similar to mature neutrophilexcept the nucleus is bi-lobed.

Numerous bright orange spherical granules

Basophil10-15 m

Similar to mature neutrophil.Large purple-black

granules that may obscure the nucleus


CELL TYPE

& SIZE

NUCLEUS COLOR,TEXTURE, & SHAPE

CYTOPLASMIC COLOR

Lymphocyte7-15 m

Mononuclear with a high N/C ratio. Nucleus is oval, round,

or slightly indented. Chromatin is diffusely dense

or coarse with clumpy masses. Nucleoli are non-

visible. A small pale chromocenter may be present.

Pale or medium blue, translucent, and may

contain several azurophilic granules

AtypicalLymphocyte

10-25 m

Irregularly shaped or unusual chromatin pattern. Nucleus may be stretched like a band across the cell. The nucleus may have visible nucleoli.

Abundant cytoplasm with vacuoles; radial and peripheral basophilia; increased number of azurophilic granules.

Plasma Cell10-20 m

Round to ovoid, usually eccentric; may be binucleated; rarely multinucleated. Chromatin is coarse and clumped with dense masses arranged in a wheel-like pattern. Low N/C ratio.

Medium to deep blue and occasionally flame color with a prominent peri-nuclear clear area or hof; agranular; may have vacuoles of varying size.

Monocyte12-20 m

Mononuclear; usually convoluted or horseshoe-shaped; evenly distributed pale and delicate chromatin.

Usually abundant, dull gray- blue; vacuolated; sparse lilac granules

6.4.8. Neutrophilic Inclusions – Various neutrophil inclusions can occur for a variety of reasons. Scan a minimum of 10 fields using the 100X oil immersion objective. Tally the number of each inclusion classification per field and report as follows:

Döhle BodiesToxic GranulationAlder’s AnomalyChédiak-Higashi AnomalyAuer bodiesVacuolesPhagocytized materialReview the Neutrophils in a minimum of 10 fields using the 100X oil immersion objective. Refer to appendix 4 for a brief summary of the various inclusion appearances and morphology, using Wright Giemsa Stain, and the hematopoietic cells involved. Tally the number of each classification per field and report as follows:


Neutrophilic Inclusions1+ 2+ 3+


6.4.9. Dysplastic Features– The following list of abnormalities can occur in the following cell types:Granulocytes:AgranularAbnormally large granulesHyposegmentation (bi-lobed, pince-nez or mononuclear)Hypersegmentation (≥6 lobes)Monocytoid changeMonocytesGranulocytic changeHyperlobulationIf any of the dsyplastic features are present, tally the number of each classification per field and report. Provide the number and type of cells as a percentage of 100 cells. For example, if 4 hypersegmented neutrophils are identified during the 100 cell differential count, report “4% Hypersegmented neutrophils present” and include the 4 cells in the neutrophil count for the 100 cell differential cell count. Repeat for each dysplastic feature noted above.

6.5.0. Spurious result identification – Engage the 40X oil immersion objective and review the slide for the any of the abnormalities identified below.

6.5.1. Agglutination – This occurs as cells aggregate into random clusters or masses when exposed to various red cell antibodies. It can be observed in certain hemolytic anemias, atypical pneumonia, staphylococcal infections and trypanosomiasis..

6.5.2. Rouleaux Formation – This is observed in the normal examination area. The erythrocytes appear in short or long stacks resembling coins or flat plates. Rouleaux occurs as a result of abnormal RBC arrangement due to biconcave surface opposition. It is commonly seen in multiple myeloma and hyperproteinemia. Review the hemogram indicies for rule of three acceptability. If the indicies are within range, rouleaux formation can be reported as present. If they are not appropriate corrective action must.

6.5.3. Hemolysis – Mild hemolysis is frequently undetected in most CBC tests. Severe hemolysis usually manifests with increased platelet counts that cannot be confirmed by the peripheral smear examination. Reject hemolyzed samples and refer to the Spurious Results SOP for further guidance.

6.5.4. If there is considerate difference between the RBC indicies and blood smear presentation, prepare and evaluate a new blood smear.


6.6.0. Platelet Estimation6.6.1. Perform a platelet estimate on any differential or RBC Morphology review

smears. This is done to correlate the automated platelet count with the estimate and check the smear for giant platelets, clumps or platelet satellitism.To do this, locate an area of approximately 150 red blood cells. The cells should be just touching or barely overlapping.Count all the platelets in that area.Follow the above step until 10 fields have been counted. Keep the total number of all 10 areas counted. Divide the total number by 10 to obtain the average platelet per field, then multiply by (x) 20,000 to obtain the platelet estimate. Refer to Calculation Section of this SOP for example.If the platelet count from the instrument does not correlate with the platelet estimate on the smear; the instrument count, the smear, and the platelet estimate process will be repeated.Occasionally platelets will be satellite around the neutrophils, giving a halo or a platelet clumping appearance. This is due to thrombocytopenia or micro fibrin clotting. 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".Notify the Ward/OPD, Clinic or Physician that the platelet count is inaccurate. Request new EDTA and Citrate tubes for evaluation Document that "Platelet count inaccurate due to platelet clumping or platelet satellitism present. Another specimens requested for evaluation".

6.6.2. If a significant numbers of giant platelets and/or platelet clumps are detected, a peripheral estimate of WBCs should be done to prevent reporting spuriously high white blood cell count.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.

Platelets Between 2-3 m in diameter

Cytoplasmic fragments with red-purple granules and no nucleus

Megakaryocytes Between 3-6 m in diameter

Usually round, containing multiple red-purple granules

6.7.0. Calculations: 6.7.1. WBC Correction for NRBC or Megakaryocyte presence - If 10 or more

NRBCs or Megakaryocytes are present use the below formula: WBC count X 100

= Corrected WBC Count # NRBCs or Megakaryocytes + 100


example: 20 X 100

WBC count = 20 X 10/L = 18.2 X 10/L Corrected WBC NRBCs = 10 10 + 100

Document that the WBC count was corrected due to NRBCs or Megakaryocytes present.

6.7.1. To perform platelet estimation, follow the below example:Platelet estimate = Average of platelets in 10 field counted X 20,000

For example: Total of 10 field counted = 100 platelets Average platelet count = 100 / 10 = 10

Platelet estimate = 10 X 20,000 = 200 X 10/L

Peripheral estimation of the WBCs should be done to check the accuracy of the actual count. To do this, follow the below steps:This estimate is done on 100X oil immersion where the red blood cells are just beginning to touch each other. Count the number of WBCs in ten (10) fields of a stained smear. Take the average of this count and use the following table:

6.8.0. Interpretation & Reporting Results:

After the 100 cells count, RBC morphology and platelet estimation is completed, reviews the results and report.

Note: Pyknotic cells: The nuclear chromatin condenses into a solid, structureless mass. THEY ARE NOT COUNTED IN THE DIFFERENTIAL. They are usually found in old blood i.e., from outlying clinics. Outlying clinics are required to submit smears they have prepared from fresh specimens when requesting CBCs through this laboratory.

Smudge or Basket cells: Cells that are obviously white blood cells in origin, resulting in a loss of characteristic shapes and forms thereby precluding identification of the cells. An increased number of these cells are seen in lymphocytic leukemia. If necessary prepare a new blood film using a 1:5 ratio of 22% albumin to whole blood. This will markedly reduce smudge formation and aid in diagnosis. Smudge or basket cells are reported as Present. Refer any unusual smear to the supervisor and/or the pathologist. Peripheral smears will be kept for seven (7) days to assure further .


verifications of the smear if requested by the physician. Abnormals kept longer

6.9.0. Sources of error:Certain cell types, particularly eosinophils and Basophils, may be distributed in a nonrandom manner on a blood film. High percentages of these cell types, especially if found in a limited area of the film, should be rechecked before the numbers are reported.

6.10.0. Limitation of the procedure:Although morphologic evaluation of a stained blood film is one of the most important hematologic procedures, the manual differentiation of leukocytes is an inaccurate and un-reproducible method.It is subject to errors that cannot be eliminated such as sampling errors, uncontrollable errors in cell distribution on the blood film, and human inconsistency in cell interpretation.

7.0. REFERENCES:7.1.0. Reference Leukocyte Differential Count, NCCLS Document H20-A, Vol.

12 No. 1, March 1992.7.2.0. Brown, Barbara A., Hematology: Principles and Procedures, Lea and

Febiger Book Publisher, Sixth Edition, 1993, Pages 102 to 105.7.3.0. Lee, Richard G., Windrobe’s Clinical Hematology; Lea and Febiger Book

Publisher, Ninth Edition, 1993, Pages 223 to 238.7.4.0. Hoffman, Ronald, Hematology: Basic Principles and Practice, Second

Edition, Churchill Livingston Inc., 1995, Pages 308 to 312.

8.0. DISTRIBUTION LIST

Section/Area No. issued Date Issued Sign.Quality ManagementFront office/DispatchSpecimen ManagementHematology SectionClinical ChemistryBlood BankMicrobiologyHistology & Cytology


9.0. TRAINING LOG

Document read by Signature Date


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