Analysis SOP by liaoqinmei

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									Institution                                                                    Code:
Laboratory name             Standard Operating Procedure (SOP)                 Version: no.
Location                                                                       Date: of release
Head/Responsible person   Digestion-decontamination of specimens               Page: 1 of 6
                                    using NALC-NaOH

  1. Digestion-decontamination of specimens using NALC-NaOH



  2. Objectives and scope

  This SOP describes the procedure of Decontamination, Digestion and LJ culture
  performance. N-acetyl-l-cysteine (NALC) acts as a mucolytic agent to liquefy sputum.
  The NaOH in a final concentration of 1.5% will decontaminate the specimen. Sodium
  citrate, by chelating heavy metal ions present in the specimen, will exert a stabilizing
  effect on the NALC. The phosphate buffer will neutralize the NaOH to prevent loss of
  mycobacterium in the specimen, which occur after prolonged exposure to the NaOH.

  This SOP is applicable to all employees of the laboratory.


  3. Abbreviations, definitions and terms

       •   CPC      Cetylpridinium chloride
       •   LJ       Löwenstein Jensen
       •   NALC     N acetyl L cysteine
       •   NaOH     Sodium Hydroxide
       •   N.A.     Not applicable
       •   QC       Quality Control
       •   SOP      Standard Operating Procedure


  4. Tasks, responsibilities and accountabilities

      Task                           Responsible                     Accountable
      Preparation of Reagents        Lab personnel in Reagent        Laboratory manager
      used in decontamination        preparation section
      Reconstitution of              Lab tech in decontamination     Laboratory manager
      decontamination mixture        section
      Decontamination of samples     Lab tech in decontamination     Laboratory manager
                                     section
      Preparation of smears          Lab tech in decontamination     Laboratory manager
                                     section
      Inoculation of MGIT tubes      Lab tech in decontamination     Laboratory manager
                                     section
      Inoculation of LJ slants       Lab tech in decontamination     Laboratory manager
                                     section


  5. Safety and environment

  •    For safety measures use Personal Protective clothing and equipment (gloves,
       Biosafety cabinet class II etc) must be used when performing this procedure.
  •    In case of biohazard incident/Accident, refer to SOP X “Biosafety Manual”
  •    Waste disposal
       All contaminated materials must be placed in biohazard bags/containers during
       working. No contaminated waste should be thrown on the floor.


  Source: GLI Stepwise Process towards TB Laboratory Accreditation
Institution                                                                  Code:
Laboratory name            Standard Operating Procedure (SOP)                Version: no.
Location                                                                     Date: of release
Head/Responsible person   Digestion-decontamination of specimens             Page: 2 of 6
                                    using NALC-NaOH

      All contaminated material will then be autoclaved, re-usable material cleaned and
      disposable material incinerated.


  6. Procedure

  6.1 Sample

  The specimens collected are mainly sputa.

  Urine specimens should be centrifuged for 30 minutes at 3000 g. The supernatant is
  discarded and the sediment is treated as sputum.

  Specimens from sterile sources (aspirates such as CSF) may be planted directly on
  culture medium; they do not need to be decontaminated.

  6.2 Reagents and Materials

      •   50 ml sterile conical tube
      •   Balance sensitive to 0.1g
      •   BioSafety Cabinet class II
      •   Sterile cryovials
      •   Calibrated sterile plastic Pasteur pipette
      •   Disinfectant ( e.g. phenol, Lysol)
      •   High speed refrigerated centrifuge ( 3000 g) at 4oC equipped with protective
          shields and 50 mL conical tube adaptor
      • Incubator
      • LJ plain media
      • N acetyl L cysteine
      • Sterile PBS pH 6.8
      • Sterile 2.9% sodium citrate
      • Sterile 6% hydroxide sodium **
      • Timer
      • Conical Flask 250ml
      • 2000ml Discard Jar
       ** Concentration of NaOH may be reduced to 4% if needed


  6.3 Detailed Stepwise Procedure

  6.3.1 Preparation of the digestion solution (200 mL).

  1. Mix 100 mL of 6 % sterile sodium hydroxide (refer to SOP ‘Preparation of 6%
     NaOH”) to 100 ml of 2.9% sterile sodium citrate (refer to SOP “Preparation of
     the 2.9% sodium citrate”).
  2. Add 1 g of NALC to the mixture and mix well.
  3. This is the 200 ml of NALC-NaOH digestion-decontamination mixture ( for
     treating approximately 10 samples).

  6.3.2 Calibration of the sterile plastic Pasteur pipette.




  Source: GLI Stepwise Process towards TB Laboratory Accreditation
Institution                                                                  Code:
Laboratory name            Standard Operating Procedure (SOP)                Version: no.
Location                                                                     Date: of release
Head/Responsible person   Digestion-decontamination of specimens             Page: 3 of 6
                                    using NALC-NaOH

  1. Dispense into some recipient (e.g. glass tube) exactly 1 ml of distilled water.
  2. Using a sterile plastic Pasteur pipette of the same type that will be used in
     routine, aspirate the complete 1 ml of distilled water.
  3. Dispense the water, drop per drop, as homogeneously as possible and count the
     number of drops.
  4. Repeat the operation 5 to 10 times with a new sterile plastic Pasteur pipette.
  5. Calculate the average and divide per 10: the number obtained is approximately
     the number of drops needed to dispense 0.1 ml.

           OR

  1. Using a sterile plastic Pasteur pipette of the same type that will be used in
     routine, aspirate distilled water
  2. In the balance, dispense and count drop per drop the distilled water until the 0.1
     g is reached (0.1g corresponds to 0.1 ml of water).

  6.3.3 Digestion/decontamination

  Note:
  If the specimen volume exceeds 10 ml, digest to homogenize and divide the sample
  into two equal parts. Proceed with the two resultant samples separately for
  neutralization, then after processing pool the sediment for inoculation.

  1.  Label MGIT tubes, LJ paired slants and slides with laboratory numbers.
  2.  Work in a BSC II which is working properly and is certified.
  3.  Do not decontaminate/process more than 16 samples at a time.
  4.  Arrange samples on the rack in ascending lab number.
  5.  Judging from the volume of each sample, carefully pour an equal volume of the
      digestion mixture from the pre-filled falcon tube.
  6. Liquefy sputum by vortexing for 10 seconds until specimen is homogenize.
  7. Start timer for 20 minutes as soon as the first drop of the digestion mixture is
      added to the first specimen.
  8. After 5 minutes, vortex for 10 seconds again.
  9. Thoroughly mix the contents of conical tubes to ensure that the inside surfaces
      have been well decontaminated (including the inner portion of cover).
  10. Let stand for more 15 minutes, for a total time of contact of 20 minutes.
  11. Add sufficient volume of phosphate buffer to achieve a total volume of 45 ml. DO
      NOT touch the centrifuge tube with the buffer container when dispensing the
      buffer.
  12. Mix solution by inverting each tube several times, load centrifuge carriers in
      safety cabinet.
  13. Centrifuge for 15 minutes at 3000  g. in sealed, domed centrifuge carriers.
      (Note: Carriers must balance in the centrifuge)
  14. Open carriers in the biological safety cabinet. Check for broken centrifuge tubes.
  15. Pour supernatant off into a splash proof container containing 30 ml of pure lysol
      in a 2000 mL discarding jar ( final concentration of Lysol is at least 2 %).
  16. Leave approximately 2-5 ml of sediment, depending on the test to be run.
      Resuspend the sediment by gentle vortexing.
  17. Using a calibrated pipette aspirate 500ul of the sediment, and inoculate the pre-
      labeled MGIT tubes, if required.




  Source: GLI Stepwise Process towards TB Laboratory Accreditation
Institution                                                                    Code:
Laboratory name            Standard Operating Procedure (SOP)                  Version: no.
Location                                                                       Date: of release
Head/Responsible person   Digestion-decontamination of specimens               Page: 4 of 6
                                    using NALC-NaOH

  18. Before inoculating on any of the slopes, aseptically pour off all the water of
      condensation (via the glass side) in the LJ slant then inoculate the pre-labeled
      paired plain LJ media with approximately 0.1 ml using a sterile plastic Pasteur
      pipette.
  19. Tilt the tubes in order for the inoculum to spread all over the slant. Leave the
      slopes in a tilted position allowing the inoculum to be spread all over the surface
      of the slant.
  20. Aseptically draw a portion of sediment and make a standard smear for
      microscopy of 1cm by 2 cm in area, on the pre-labeled slides and place them on
      the slide warmer for drying at 65oC for 2 hours.
  21. Store the remaining sediment at -20ºC or below as a back up for contaminated
      inoculated LJ slants (up to 4 weeks) or other repeat test.
  22. Discard the remaining PBS (Do not reuse).
  23. Discard the pipette in the or biohazard bag.
  24. Place the tilted slants in the incubator at at 37°C till it dries up for 2 days, and
      put them back in a up-right position.
  25. Incubate the inoculated Lowenstein Jensen media slants in a chronological order
      in the incubator at 37C.
  26. Fill out:
              a. Sample Processing and Culture Worksheet, Appendix 1.
              b. MGIT inoculation worksheet
              c. LJ Primary culture Register book.

  6.4 Quality Control
  1. Keep a monthly record of :
         a. Contamination rate: the acceptable range is 2-5%. Contamination rate
            is obtained by dividing the total amount of contaminated slopes by the
            total amount of slopes cultured on a monthly basis. <2% indicates harsh
            decontamination, which means that too many tubercle bacilli are killed.
            On the other hand, a too high contamination rate may indicate problem
            with the specimen, for example.
         b. False negative culture ( acceptable range is 2-5% ): is obtained by
            dividing the total amount of new smear positive cases that give negative
            culture by the total number of new smear positive cases. This rate may
            give information on the decontamination phase as well as on the
            microscopy.
  2. If the laboratory is experiencing delays in delivery of specimens the
     contamination rate may be greater than 5%. If a rate of 5% persists, ensure that
     specimens are completely digested, since partially digested specimens may not
     be completely decontaminated.
  3. In case of delaying specimen (transit time more than 4 days), consider the use of
     cetylpyridinium chloride (CPC).
  4. Interpreptation of QC Indicators:
            Contamination rate > 5%
                       Poor final concentration of the NaOH
                       Specimen delay more than 4 days at RT
                       Sputum container dirty/ improper
            Contamination rate <2%
                       Too high final concentration of NaOH
                       Too much exposure to decontaminant
            False negative culture rate > 5%



  Source: GLI Stepwise Process towards TB Laboratory Accreditation
Institution                                                                  Code:
Laboratory name            Standard Operating Procedure (SOP)                Version: no.
Location                                                                     Date: of release
Head/Responsible person   Digestion-decontamination of specimens             Page: 5 of 6
                                    using NALC-NaOH

                          Too harsh decontamination
                          Poor microscopy ( too high false positive smear)
               False negative culture rate < 2%
                          Poor decontamination with fresh specimen
               Positive culture from negative smear rate
                          Plot the rate every month and check for any changes: increase
                          could be due to poor microscopy (false negative smears).


  7. Related documents

  SOP “Preparation of Phosphate Buffered Saline (PBS) solution pH 6.8”
  SOP “Preparation of 6% Sodium Hydroxide solution”
  SOP “Preparation of 2.9% Sodium Citrate”


  8. Related forms

  N.A.


  9. References

  N.A.


  10. Attachments / Annexes

  Appendix 1, Sample Processing and Culture Worksheet




  Source: GLI Stepwise Process towards TB Laboratory Accreditation
               Sample Processing and Culture Worksheet

Tech. Name:                                              Date:

Number of samples processed:

Reagent Information
     Reagent                  Batch #              Date Prepared                   Expiration Date
6% NaOH
2.9% Sodium Citrate
NALC
LJ Media
PBS (Buffer)


Fill out the table completely
                 Sample              MGIT                          Slides
Sample                                (tick if   LJ Media
               Laboratory                                         prepared           COMMENT
     #                                done)      (tick if done)
                                                                  (tick if done)
                 Number
     1

    2

    3

    4

    5

    6

    7

    8

    9

   10


Incubation Start date: __________ Time: ________ Tech Signature: ___________

Reviewed by:_______________           Date: ____________




Source: GLI Stepwise Process towards TB Laboratory Accreditation

								
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