Microscopy QA_TB 04-03_V1.0.doc
Table of contents
1. PURPOSE 2
2. SCOPE 2
3. RESPONSIBILITY 2
4. CROSS-REFERENCES 2
5. PROCEDURES 2
5.1 Preparation of Positive and Negative Quality Control Slides 2
5.2 Quality Control Testing of Staining Solutions 3
5.3 Daily Quality Control 4
5.4 Internal Quality Assurance Indicators 4
5.5 Blinded re-checking of slides 4
5.6 External Quality Assurance (EQA) 6
6. REFERENCES 6
7. CHANGE HISTORY 6
1. PURPOSE
This operating procedure describes the procedures to be followed for quality assurance of microscopy for detection of tuberculosis in the ______TB Laboratory.
The procedure covers (a) quality control testing of staining reagents for Ziehl-Neelsen and fluorescent staining, (b) internal QC indicators, (c) procedure for blinded re-checking of slides.
2. SCOPE
This procedure covers the quality assurance protocols to be put in place to assure the quality of microscopy performed in the ______TB Laboratory.
3. RESPONSIBILITY
The Head of ______TB Laboratory is responsible for implementation of this procedure. The Head of the ______TBLaboratory may delegate responsibility for supervision of the procedure to a suitably qualified member of the laboratory staff.
All staff members working in the ______TB Laboratory are responsible for carrying out the activities as described herein. All users of this procedure who do not understand it or are unable to carry it out as described are responsible for seeking advice from their supervisor.
4. CROSS-REFERENCES
See: / Document Matrix_TB 01-01_V1.0.docLocation:
Refer to SOPs listed under TB 02 (General Procedures), TB 03 (Specimen Handling), TB 04 (Microscopy Methods) and TB 07 (Equipment Use and Maintenance).
5. PROCEDURES
5.1 Preparation of Positive and Negative Quality Control Slides
· Prepare batches of positive and negative control slides from suitable sputum specimens. Prepare at least 20 smears of each, as identical in size and thickness as possible, giving each series the same QC identification number.
· Specimens must have been thoroughly examined (five slides prepared and 100 fields of each slide examined by at least two readers),
· A negative control and a low positive control (1+, 10-99AFB /100 fields) should be selected, and homogenized by standing overnight at room temperature.
· After smear validation and preparation of the batch of slides, check two slides from each batch after staining, and note the average number of AFB for the 1+ on the QC Slides Preparation Form.
Use: / QC Slides Prep_form.docLocation:
· Store slides in appropriately labeled slide boxes, with date of preparation recorded.
5.2 Quality Control Testing of Staining Solutions
· Check every newly prepared staining solution with unstained control slides, using at least one positive (1+) slide and one negative slide.
· Stain the positive smear(s) once according to SOPs.
See: / ZN Microscopy_TB 04-01_V1.0.docFluorescence Microscopy_TB 04-02_V1.0.doc
Location:
· Stain negative slides three times in total to make sure any contaminants present in decolouriser or counterstaining solution will be visible.
· Examine the controls and note the results in the Quality Control of ZN Stains or Quality Control of Auramine Stains, under the batch number (and/or preparation date) of the new solutions.
Use: / QC ZN Stains_form.doc ORQC Auramine Stains_form.doc
Location:
· Unacceptable control results for ZN can include:
a. Positive control AFB are not stained bright pink or red, or are too few in number.
b. Negative control remains bright pink or red after decolourisation.
c. Background is too intense or contains too many artifacts.
· Unacceptable control results for FM can include:
a. Positive control AFB are not stained bright yellow or are too few in number.
b. Negative control remains bright yellow after decolourisation.
c. Background is too dark or contains too many fluorescent artifacts.
If one or more of these outcomes occurs, check if something went wrong with the solution preparation. If this seems unlikely, repeat the procedure with two more slides from each control, paying attention to correct staining technique. If these controls have the expected results, they can be accepted.
However, if the repeat controls also show unacceptable results, discard the staining solutions and prepare new ones.
5.3 Daily Quality Control
· Include a positive and a negative control with each day's reading.
· Read control slides before patient smears; this will help with adjusting the focus and checking the proper functioning of the instrument.
· If unacceptable results described above are found, re-stain that day’s routine smears together with new controls, paying attention to correct technique; if these new controls are also unacceptable, then prepared new staining solutions and repeat staining process.
· Record results of control slides on the Ziehl-Neelsen Microscopy Worksheet or Auramine Microscopy Worksheet along with results of specimens.
Use: / Ziehl-Neelsen Microscopy Worksheet_form.doc ORAuramine Microscopy Worksheet_form.doc
Location:
5.4 Internal Quality Assurance Indicators
Monitor the overall laboratory smear microscopy performance by monthly counts and plotting a graph of:
· number of smears performed (ZN and Auramine)
· positivity rate (ZN and Auramine)
These indicators are an early warning of problems and indicate the need for corrective action. Internal monitoring can also contribute to staff motivation and self reliance. A report on monthly QA indicators will be prepared and disseminated to all laboratory staff. If any corrective actions are required an Internal Audit Report will be issued.
Use: / Internal Audit Report TB Lab_form.docLocation:
5.5 Blinded re-checking of slides
All slides must be stored in chronological date order in suitably labeled slide boxes for easy retrieval for re-checking. Blinded re-checking of slides will be carried out in by the Laboratory Quality Manager, or other designated, experienced laboratory technologist.
Re-checking will be done on a monthly basis, with four ZN and four FM slides being re-checked from each laboratory. Slides will be sampled during the first week of the month, with all slides being read the previous month being sampled.
Sampling will be performed by Quality Manager according to the following procedure:
Note: a calculation for sampling will be based on the number of slides processed by ZN and FM by the lab. Perform a separate calculation for each method (ZN and FM) and for each laboratory):
Sampling interval = Number of slides in previous month / sample size (4)
Starting point = 1 to (sampling interval – 1)
The starting point will be selected by the Quality Manager at the time of sampling.
Example: If TB Lab processes 400 FM slides the previous month, sampling interval = 400/4 = 100. Starting point can be between the 1st and 99th slide. If starting point is chosen as 3, then FM slides number 3, 103, 203 and 303 will be selected for re-checking.
The Quality Manager will record the original slide numbers selected on the Inter-laboratory Microscopy Re-checking Form (which is to be kept as confidential from all laboratory staff, with the exception of Head of the ______Laboratory). The Quality Manager will blind the specimens and designate a new code to each slide. The code will also be recorded on the Microscopy Re-checking Report.
Use: / Microscopy Re-checking Report_form.docLocation:
Four blinded ZN slides and 4 blinded FM slides will be provided to senior technologist/technician for reading. A form containing only blinded codes will be provided for the purposes of recording of results. No re-staining will be performed prior to reading of slides. Interpretation of smear results will be according to WHO / IUATLD guidelines.
See: / ZN Microscopy_TB 04-01_V1.0.docFluorescence Microscopy_TB 04-02_V1.0.doc
Location:
Results will be recorded on the following form:
Use: / Microscopy Re-checking Results_form.docLocation:
The Quality Manager will request results to be provided on the designated form within a maximum four days and will compile reports of the re-checking once all results have been reported.
The Head of the ______TB Laboratory and the Quality Manager will jointly review results and discuss with all laboratory staff involved in microscopy procedures in the laboratory. Corrective and preventive measures will be implemented and documented by the Quality Manager.
5.6 External Quality Assurance (EQA)
The TB Laboratory will participate in an appropriate microscopy external quality assurance scheme, by an accredited international organization. Such schemes provide panels for microscopy, usually 2-3 times per year.
6. REFERENCES
World Health Organisation. Laboratory Services in Tuberculosis Control. Part II. Microscopy. WHO/TB/98.258
7. CHANGE HISTORY
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