Pro6.4-B-04 Wrights Stain -Diff Quick- Manual Method by lsy121925

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									                           Penny Stevens                                  Procedure Number           Date
Author(s), Name & Title
                              International QA/QC Coordinator               Pro64-B-04 Version 1       Jan 06
SMILE 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.

Copy _______ of ________                                              Effective Date:



                                                   YOUR LAB



HEMATOLOGY SECTION


C. DIFFERENTIAL CELL COUNTS

C.2. WRIGHT’S STAIN MANUAL METHOD

C.2.1. PRINCIPLE:

        Wright’s stain is a polychromatic stain consisting of a mixture of eosin and methylene
        Blue. When applied to blood cells, the dyes produce multiple colors based on the ionic
        charge of the stain and the various components of the cell. The eosin ions are negatively
        charged and stain basic cell components an orange to pink color. The methylene blue
        ions are positively charged and stain the acid cell components in varying shades of blue.
        The neutral components of the cell are stained by both components of the dye producing
        variable colors.

C.2.2. SPECIMENS

        1. Thin wedge blood smears are prepared on pre-cleaned glass slides from freshly drawn
           capillary or venous blood anticoagulated with EDTA.

        2. The blood smear slide must be properly prepared, labeled and completely dry before
           commencing with the staining procedure.

        3. For best results, prepare and stain smears within two (2) to three (3) hours of
           specimen collection.

C.2.3. EQUIPMENT AND REAGENTS:

        1. EQUIPMENT:

             1.1. Four (4) coplin jars



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        1.2. Pre-clean glass slides

        1.3. Whatman Grade 41 filter paper – 110mm diameter

        1.4. Funnel – short neck

     2. REAGENTS:

        2.1. Diff Quick Fixative Solution

        2.2. Diff Quick Solution I (Red)

        2.3. Diff Quick Solution II (Blue)

        2.4. Distilled Water

        NOTE: The reagents are flammable and must be stored in the flammable cabinet.
        Keep only the minimum working supply, appropriately labeled with hazard warnings,
        in the section.

C.2.4. PROCEDURE:

     1. Dip the blood smear into the Diff Quick fixative solution five times for one (1)
        second each time. Wipe the back of the slide with gauze to prevent carry over into
        the next stain. Do not allow the fixative to dry on the slide as this will result in an
        over-fixed wet appearance. Immediately proceed to the next step while the slide is
        still wet.

     2. Dip the fixed blood smear into the Diff Quick solution I five times for one (1) second
        each time. Wipe the back of the slide with gauze to prevent carry over into the next
        stain. Do not allow the stain to dry on the slide. Immediately proceed to the next step
        while the slide is still wet.

     3. Dip the fixed blood smear into the Diff Quick solution II five times for one (1) second
        each time. Wipe the back of the slide with gauze to prevent carry over into the next
        stain. Do not allow the stain to dry on the slide. Immediately proceed to the next step
        while the slide is still wet.

     4. Rinse the fixed blood smear with distilled water five times for one (1) second each
        time. Wipe the back of the slide to decrease drying time. Set the slide upright to air
        dry. Allow the slide to dry thoroughly before reading.

     5. To decrease drying time, slides may be blown dry. Set the blow dryer on cool and
        hold the slide at least 12 inches from the air flow until completely dry.



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C.2. WRIGHTS STAIN MANUAL METHOD
C.2.5. PROCEDURAL NOTES:

     1. Properly stained smears will display the following characteristics when examined
        microscopically:

        1.1. Red to buff pink colored red blood cells.

        1.2. White blood cell nucleus coloration:

              1.2.1.     Lymphocyte and Neutrophil nucleus are dark purple.
              1.2.2.     Monocyte nucleus is light blue to purple.

        1.3. White blood cell and platelet cytoplasm appearance:

              1.3.1.     Neutrophil cytoplasm is light pink with lilac granules.
              1.3.2.     Lymphocyte cytoplasm varies from light to dark blue.
              1.3.3.     Monocyte cytoplasm is gray-blue with few fine reddish granules.
              1.3.4.     Basophil cytoplasm is clear with few blue-black granules.
              1.3.5.     Eosinophil cytoplasm is pink to clear with many large red to orange
                         granules.
              1.3.6.     Platelets appear as light blue to colorless with red violet granules.

              NOTE:        The Diff Quick Fixative is insufficient to adequately fix the
              basophil granules. As a result, most of the granules are lost in the staining
              process and the basophils appear ‘agranular’, which can result in
              misidentification. This basophil appearance is inconsistent with images
              presented in current literature.

        1.4. Precipitation: Rare to slight precipitation is acceptable. As stain solution II
             evaporates, excess precipitation will form. Filter the stain solution II as needed
             to remove excess precipitation.

        1.5. Contamination: Bacterial or semen contamination can occur as a result of shared
             use between microbiology and hematology. If either is present in the stain
             solution, replace all reagents and perform maintenance as indicated in section
             C.2.7.

     2. Sources of error:

        2.1. Fixation:

              2.1.1.     Inadequate fixation may result in a detached specimen, indistinct
                         nuclear detail or poor granule staining. This is especially evident in
                         basophils and primary myeloid granules.



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             2.1.2.     Dry freshly prepared smears for at least fifteen (15) minutes to prevent
                        drying artifacts. Use only pre-cleaned glass slides. Slides other than
                        pre-cleaned may result in improper fixation and the presence of haze
                        over the slide due to acid-base disturbance. Pre-cleaned slides are
                        necessary for quality stain appearance.

             2.1.3.     Use only anhydrous methanol for fixative. Keep it tightly closed and
                        away from moisture or chemical fumes.

             2.1.4.     If fixation problems occur or increased granularity visualization is
                        necessary, increase the fixation period to one (1) minute.

       2.2. Staining:

             2.2.1.     Excessively blue stain appearance may be due to improper smear
                        preparation, prolonged staining time or increased stain alkalinity.

             2.2.2.     Excessively pink stain appearance may be due to insufficient or
                        decreased staining time, prolonged washing time or increased stain
                        acidity.

       2.3. Rinse:

             2.3.1.     Use only distilled water or phosphate buffered saline for the final rinse.
                        Tap water is never acceptable as a substitute for the rinse since it can
                        disturb the acid base balance and may contain chlorine.

             2.3.2.     Vigorous or prolonged washing may dislodge cell from under-fixed
                        smears, cause nuclear clumping or under-staining.

             2.3.3.     Insufficient rinsing may result in a blue smear or excessive precipitate.

       2.4. Drying: Air drying is the preferred method. Forced rapid drying may alter the
            color intensities by shortening the exposure time to the wash water because the
            red spectrum of colors continues to develop as long as the cellular elements are
            wet.

C.2.6 QUALITY CONTROL

    1. Prepare one differential slide daily using a patient sample with a normal MCV, MCH,
       MCHC and total white count. Stain the slide as indicated in procedure section C.2.4.
       Review the slide for Color, Precipitation and Contamination.

    2. If the color does not meet the specifications identified in the SOP as indicated above
       or precipitation and/or contamination are present, the quality is determined to be

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        unsatisfactory. Indicate unsatisfactory on the Quality Control (QC) form and replace
        the stain as indicated in the following section. Document the problem and corrective
        action in the appropriate section of the QC form. If the stain is determined to be
        satisfactory, indicate satisfactory on the QC sheet.



C.2.7. MAINTENANCE

     1. Perform maintenance weekly or as needed for problems indicated in previous
        sections.

     2. Discard reagents into the gram stain hazardous waste containers located in the
        microbiology department.

     3. Clean the coplin jars using mild soap and tap water. Perform a final rinse using
        distilled water and allow the jars to air dry.

     4. Use two sets of coplin jars and rotate the sets weekly allowing the jars to dry
        thoroughly before use.

     5. Replace the fixative, stain and water into the appropriately labeled jars with accurate
        hazard warning labels. Filter the stain solution II (blue stain) using a short neck
        funnel and 110 mm diameter filter paper folded into a cone.

     NOTE: The gravimetric filtration process is slow – do not attempt to speed it up or it will
     force particulates into the filtered stain.

B.2.8. APPENDICES:

     1. Diff Quick Maintenance / Quality Control Form.

     2. SOP Validation Form and SOP Change Control.

     3. SOP Approval.

B.2.9. REFERENCES:

     1. Brown, Barbara A.; Hematology: Principles and Procedures, Sixth Edition, Lea and
        Febriger Book Publisher, 1993, Pages 97-100.

     2. Hoffman, Donald; Hematology: Principles and Practice, Second Edition, American
        Book Publisher, 1995, Pages 2202-2203.

     3. Carr, Jacqueline H. and Rodak, Bernadette F.; Clinical Hematology Atlas, W.B.

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C.2. WRIGHTS STAIN MANUAL METHOD
       Saunders Company, 1999, Pages 33-86.

    4. Laboratory CQI SOP, 8 April 2004.




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                                        SOP VALIDATION
SOP NAME:
C.2. WRIGHT’S STAIN MANUAL METHOD

Clear and specific title and principle: yes / no
Comments:


All necessary supplies, equipment, and materials are listed: yes / no
Comments:


SOP is sufficiently detailed to be understood but not overly complex: yes / no
Comments:


SOP text adequately describes process/procedure: yes / no
Comments:


SOP accomplishes purpose: yes / no
Comments:


Reviewed by: (Name & Title)

Signature: __________________________                       Date: __________________


SOP CHANGE CONTROL

Date                           Change                       QA          OIC      Med Dir




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                                       SOP APPROVAL

                                              SIGNATURE                DATE
        PREPARER

    QA COORDINATOR

    LABORATORY OIC

   MEDICAL DIRECTOR



                                       ANNUAL REVIEW

REVIEWER SIGNATURE              DATE         REVIEWER SIGNATURE               DATE




DOCUMENT COPY CONTROL               DATE: ___________     # COPIES __________
LOCATIONS
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                                2                                        10

                                3                                        11

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SUPERSEDES: _21 Jan 1998_____

DATE SOP RETIRED: __________




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