Title: - Welcome to Project pSMILE! — pSMILE Portalresources.psmile.org/resources/process-control/section... · Web viewIn cases of microcytic / hypochromic, the rule of 3 should

Botswana-Harvard HIV Reference

Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284

Standard Operating Procedure

Manual Differential Cell Count

Document: BHHRL/005PR01

Version: 1

Author: See below Document Number: Pro64-B-11Effective (or Post) Date: 17-Jan-09

Document Origin:BHHRL Company: NASMILE Approved by: Heidi Hanes

Review by Heidi Hanes Review date 7 Feb 2012SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering their use in other applications. If you have any questions contact SMILE.

___________________________________________________________________________________________ Effective Date: 01/06/2008 Page 1 of 17

Confidential Controlled Document


Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

Title: Manual Differential Cell Count

Prepared By:Name, Title Signature, Date

Munyaradzi P. Mangwendeza, QA ManagerMatshediso Zachariah, Acting Deputy QA

Manager

Approved By:Name, Title Signature, Date

Dr. Rosemary Musonda, Laboratory Director

Annual Review

Review Date Revision Date Signature, Date

Distributed To:

Location: Copy Number: Location: Copy NumberMaster file 1Director –

Dr. Musonda2

Director –Dr. Mine

3

Lab Managers – Mr. S. Moyo

Mrs. K. Makhaola

4

5Lab

Coordinator6

Copy Number: _________

1. General Policy1.1. Manual differential count should be done on all the samples that did not give the

automated differential count analyzed from the automated analyzer.____________________________________________________________________________________________________Effective Date: 01/06/2008 Page 2 of 17



Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

1.2. All the results from patient samples that are suspect to be abnormal should be analyzed under the microscope for any corrections and clarifications to be made.

2. Principle2.1. Manual differential count should be done on all the samples that did not give the

automated differential count analyzed from the automated analyzers.2.2. All the results from patient samples that are suspect to be abnormal should be analyzed

under the microscope for any corrections and clarifications to be made should also have a manual differential count done.

2.3. A high white blood cell count and shifts in the differential cell count on the automated analyzer are also indicative of the need for a manual differential cell count.

3. Purpose3.1. The purpose of this document is to establish guidelines for performing the differential

cell count manually and offer guidelines of performing other blood cell analysis and platelets.

4. Scope4.1. The SOP covers the white cell manual differential cell count procedures, red blood cell

analysis as well as platelet analysis.

5. Responsibilities5.1. It is the responsibility of all hematology laboratory personnel to prepare blood film in

samples with abnormal results from the analyzer. 5.2. The Head of Section shall be responsible for the blood film results.5.3. Hematology staff shall be responsible for the maintenance and logging of the

microscope.5.4. The Laboratory Section Supervisor shall be responsible for reviewing the microscope

log sheet every month.5.5. The laboratory section supervisor shall be responsible for entering the results derived

from the blood film into the system and file the analyzer result form.5.6. Pathologist MoH will give interpretations of results that require a pathologist expert

opinion before the results can be reported.

6. Principle: 6.1. 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

____________________________________________________________________________________________________Effective Date: 01/06/2008 Page 3 of 17



Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

abnormal blood counts. Equally important is the blood morphology, which can often reveal important diagnostic or therapy-related information.

6.2. 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.

7. Specimen: 7.1. A proper wedge peripheral blood smears stained in accordance with the May-Grunald

Giemsa Stain (a Romanowsky type blood stain) manual or automated methods.

8. Equipment and Reagents:8.1. Microscope8.2. Low power 10X objective and 50X and 100X oil immersion objectives8.3. May-Grunald Giemsa stained peripheral smear with patient identification number clearly

marked.8.4. Cell differential counter 8.5. Patient’s labeled automated FBC instrument report with histograms8.6. Immersion Oil

9. Quality Control: 9.1. Quality control is performed in accordance with the May-Grunald Giemsa Stain (a

Romanowsky type blood stain) manual or automated methods. See SOP BHHRL/005PR01 Quality Control of May-Grunald Giemsa Stain.

9.2. The following steps must be taken during differential testing to ensure uniform reporting within the lab:

9.2.1. Agree upon the definition of all terms used. 9.2.2. Circulate unfamiliar slides among the lab staff 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.

9.2.3. Establish criteria for peripheral smear review by the department supervisor and pathologist.

10. Procedure And Reporting:




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

10.1. Verify that the automated FBC report and blood smear patient identification match.

10.2. Place the stained slide (specimen side up) on the microscope stage. 10.3. The 10X low power objective. Examine the feathered edge (figure 1) of the

smear to determine blood film adequacy.

Figure 1

10.4. 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.

10.5. 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.

10.6. 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. Review all abnormally large leukocytes under 100X for appropriate classification and follow up testing.




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

10.7. 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.

11. Manual Differential Count: 11.1. Perform the leukocyte estimate using the high power (50X) oil immersion

objective. 11.2. Count the number of both intact and disrupted leukocytes in 10 microscopic

fields where the erythrocytes are partially but not completely overlapped. This is a slightly thicker area than the area where the differential will be performed. Divide the total number by 10 to establish the mean number of leukocytes per field. Multiply the mean by 3000 to determine the estimated WBC count/mm3.

11.3. If the quantitative leukocyte count/mm3 is less than 25,000 the number should agree within 20% of the automated count. If the estimate does not agree within 20%, repeat the WBC count on the instrument and/or prepare another smear to ensure that the correct sample was tested and/or used to prepare the smear. This method is less reliable when the automated leukocytes count/ mm3 is greater than 25,000.

11.4. Perform the 100 cell differential count. 11.4.1. Select the optimal counting area. This is the area where the erythrocytes are

adjacent but do not overlap. The 50x 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.

11.4.2. 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 in figure 2.

Figure 2.




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

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

11.4.2.2. If the automated WBC count is less than 2.0 x 10^3/uL, a 50 cell count can be performed and converted to 100% by multiplying all values by 2. Add the following comment in result slip: 50 cells counted during the manual differential and converted to percentage.

11.4.2.3. If the automated WBC count is greater than 30.0 x 10^3/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 in the result slip: 200 cells counted during the manual differential and converted to percentage.

11.4.2.4. If nucleated red blood cells (NRBC) or Megakaryocytes are seen during the differential count, enumerate them separately from the white blood cell count.

11.4.3. 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:

11.4.4. Enter the manual differential count in the result slip.

11.5. Calculate absolute cell counts using the manual differential.11.5.1. 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.

11.5.2. Convert the total percentage to decimal, i.e. 45% lymphs = 0.45. 11.5.3. Multipy the decimal by the total white count, i.e., 10.0 x 10^3/uL WBC x 0.45

Lymphs = 4.5 x 10^3/uL Lymphocytes.____________________________________________________________________________________________________Effective Date: 01/06/2008 Page 7 of 17



Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

11.5.4. 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.

11.5.5. Enter the manually calculated absolute counts from step 11.5.1 in place of the automated absolute count fields in the computer with the following comments: Absolute counts calculated based on manual differential.

11.6. If the FBC was certified, do not amend the results. Result the manual differential and enter the following in the LIS comment: Questionable automated differential results. Recommend repeat testing to verify, if clinically indicated. Absolute WBC counts (10^3/uL) calculated based on manual differential results: Enter the calculated results separated by commas.

12. RBC Morphology And WBC Review12.1. The RBC Morphology and WBC Review will be performed with every manual

differential or as a separate examination. 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:

12.2. 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:

12.2.1. Normocytic - normal size12.2.2. Microcytic - smaller than normal12.2.3. Macrocytic - larger than normal

12.3. 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 cells

12.4. Normocytic cells are not graded. Report Normocytic in LIS if microcytosis and/or macrocytosis are not present or they are present at less than 10 cells per field.




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

12.4.1. 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

12.4.2. 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:

12.4.2.1. Normochromic - normal color12.4.2.2. Hypochromic - pale color and large central pallor 12.4.2.3. Polychromasia – bluish color and usually macrocytic

12.4.3. 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

12.4.4. Normochromic cells are not graded. Report Normochromic in LIS 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.

12.5. 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.

12.5.1. Acanthocytes12.5.2. Bite Cells12.5.3. Blister Cells




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

12.5.4. Codocytes – Target Cells12.5.5. Dacryocytes – Teardrops12.5.6. Drepanocytes – Sickle Cells12.5.7. Echinocytes - Crenated or Burr Cells12.5.8. Elliptocytes or Ovalocytes12.5.9. Envelope forms or folded cells12.5.10. Schistocytes12.5.11. Spherocytes12.5.12. Stomatocytes

12.6. Review 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, & Spherocytes 1+ 2+ 3+Occasional 1% Moderate 1 – 2.5% Many >2.5%<1 to 2 cells 2 to 5 cells >5 cells

12.6.1. 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.

12.7. 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:

12.7.1. Basophilic Stippling12.7.2. Howell Jolly Bodies12.7.3. Pappenheimer Bodies12.7.4. Cabot Rings12.7.5. Hemoglobin C Crystals12.7.6. Bacteria




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

12.7.7. Parasites12.7.8. Mauer’s Dots12.7.9. Schüffner’s Dots12.7.10. Nucleus.12.7.11. Artifacts

12.8. Review 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:

RBC Inclusions1+ 2+ 3+Occasional 1% Moderate 1 – 2.5% Many >2.5%<1 to 2 cells 2 to 5 cells >5 cells

12.8.1. 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.

12.9. White Blood Cell Color

CEll Type& SIZE

NUCLEUS COLOR, TEXTURE, & SHAPE

CYTOPLASMIC COLOR

Segmented Neutrophil 10-15 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.

Banded Neutrophil10-18 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




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

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

Atypical Lymphocyte10-25 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




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

12.10. 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:

12.10.1. Döhle Bodies12.10.2. Toxic Granulation12.10.3. Alder’s Anomaly12.10.4. Chédiak-Higashi Anomaly12.10.5. Auer bodies12.10.6. Vacuoles12.10.7. Phagocytized material

12.11. Review the Neutrophils in a minimum of 10 fields using the 100X oil immersion objective. Tally the number of each classification per field and report as follows:

Neutrophilic Inclusions:1+ 2+ 3+Occasional 1% Moderate 1 – 2.5% Many >2.5%<1 to 2 cells 2 to 5 cells >5 cells

12.12. Dysplastic Features– The following list of abnormalities can occur in the following cell types:

12.12.1. Granulocytes:12.12.1.1. Agranular12.12.1.2. Abnormally large granules12.12.1.3. Hyposegmentation (bi-lobed, pince-nez or mononuclear)12.12.1.4. Hypersegmentation (≥6 lobes)12.12.1.5. Monocytoid change

12.12.2. Monocytes12.12.3. Granulocytic change

12.12.3.1. Hyperlobulation

12.13. If any of the dsyplastic features are present, tally the number of each classification per field and report comments. 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.

12.14. Spurious result identification – Engage the 50X oil immersion objective and review the slide for the any of the abnormalities identified below.




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

12.15. 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..

12.16. 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.

12.17. The rule of 3 is defined as 12.17.1. RBC x 3 = HGB 12.17.2. HGB x 3 = HCT 12.17.3. HCT x 3 = MCV12.17.4. This rule can only be applied, however, to normal erythrocytes and does

not apply when the MCV, hemoglobin, or RBC count is abnormal.12.17.5. The disagreement of "rule of three" could be seen in following situations

using automated hematology analyzer: 12.17.5.1. true abnormal erythrocytes, such as reticulocytosis, microcytosis,

hypochromic RBCs, erythrocytosis, and others; 12.17.5.2. false elevations in hematocrit, hemoglobin, or RBC counts. These may be

seen in patients with cryoproteins, lipemia, high white-blood-cell counts, and giant platelets;

12.17.5.3. instrument problem, random error may have occurred12.17.5.4. . In cases of microcytic / hypochromic, the rule of 3 should pass for

Haemoglobin and HCT but not MCV.12.18. If the indicies are within range, rouleaux formation can be reported as present. If

they are not appropriate corrective action must be taken.12.19. 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.

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

13. Platelet Estimation13.1. 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.

13.2. To do this, locate an area of approximately 150 red blood cells. The cells should be just touching or barely overlapping.



http://www.allbusiness.com/primary-metal-manufacturing/foundries-nonferrous/4503976-1.html


Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

13.3. Count all the platelets in that area.13.4. Follow step 13.2 and 13.3 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.

13.5. 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.

13.6. 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:

13.7. Values greater than 50 x 10/L: Document that "Platelet count inaccurate due to platelet clumping or platelet satellitism present".

13.8. Values less than 50 x 10/L: Notify the Clinic or Physician that the platelet count is inaccurate. Request new EDTA and Citrate tubes for evaluation.

13.9. 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.

13.10. Do manual platelet estimates counts 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

14. Calculations: 14.1. 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

For example:




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

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

NOTE:14.2. Document that the WBC count was corrected due to NRBCs or Megakaryocytes

present. 14.3. 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

14.4. Peripheral estimation of the WBCs should be done to check the accuracy of the actual count. To do this, follow the below steps:

14.5. This estimate is done on 100X oil immersion where the red blood cells are just beginning to touch each other.

14.6. Count the number of WBCs in ten (10) fields of a stained smear. Take the average of this count and use the following table:

15. Interpretations And Reporting Results:

15.1. After the 100 cells count, RBC morphology and platelet estimation is completed, reviews the results and initiated the CBC Report.

15.2. Critical Results and delta checks should be verified and reported.

16. . Procedural Notes: 16.1. Pyknotic cells: The nuclear chromatin condenses into a solid, structure less mass.

THEY ARE NOT COUNTED IN THE DIFFERENTIAL. They are usually found in old blood.

16.2. 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.

16.3. Refer any unusual smear to the supervisor and/or the pathologist.




Laboratory(BHHRL)

Tel: +267 3902671; Fax: +267 3901284




Version: 1

16.4. Peripheral smears will be kept for seven (7) days to assure further verifications of the smear if requested by the physician. Slides with abnormal findings are kept longer.

17. Sources Of Error:17.1. 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.

18. Limitation Of The Procedure:18.1. 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.

18.2. 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.

19. References:19.1. Reference Leukocyte Differential Count, NCCLS Document H20-A, Vol. 12 No.

1, March 1992.19.2. Brown, Barbara A., Hematology: Principles and Procedures, Lea and Febiger

Book Publisher, Sixth Edition, 1993, Pages 102 to 105.19.3. Lee, Richard G., Windrobe’s Clinical Hematology; Lea and Febiger Book

Publisher, Ninth Edition, 1993, Pages 223 to 238.19.4. Hoffman, Ronald, and Hematology: Basic Principles and Practice, Second

Edition, Churchill Livingston Inc., 1995, Pages 308 to 312. 19.5. Waters, Jerry, Standardization of Red Cell Morphology Reporting Video, CLE

(Clinical Laboratory Education), Milwaukee, WI.

19.6. Diggs, L.W., The Morphology of Human Blood Cells Color Atlas, Abbott Laboratories, Inc., Fifth Edition, 1985.

19.7. Glassy, Eric F., Color Atlas of Hematology, College of American Pathologists, 1998.

19.8. SOP BHHRL/005PR01 Quality Control of May-Grunald Giemsa Stain.



Documents

Title: - Welcome to Project pSMILE! — pSMILE Portalresources.psmile.org/resources/process-control/section... · Web viewIn cases of microcytic / hypochromic, the rule of 3 should