Gram stain of sputum is a valuable test for the requesting physician. It helps the physician to judge whether the patient he/she has counseled to obtain the sputum has really submitted the sputum sample or merely an oropharyngeal secretion i.e. saliva. Generally, while preparing the sputum smear, purulent part of the sputum is taken and smear is made on a clean grease free glass slide. Then the smear is left to dry in air and heat fixed. Gram stain is performed and observed using low power field of a microscope for presence of epithelial cells or polymorphoneutrophils/pus cells.

There are different criteria to judge the quality of sputum and to decide whether to accept or reject the sample.

  1. Bartlett has proposed a scheme for grading the quality of sputum specimens after a slide of the specimen had been examined at 100x magnification. He has assigned + and – values as follows:
Cells

 

Score
>25 WBC +2
10-25 WBC +1
mucus +1
>25 EPI -2
10-25 EPI -1

Any positive score (sum of + and – values assigned) is taken as an acceptable sample, 0 or – value suggests that the sample should be rejected.

 

  1. In 1975, Murray and Washington described a simple scheme for judging the quality of sputum specimens. They rejected all specimens with more than 10 squamous epithelial cells per average low-power field (LPF).

 

Average no. of EPI/LPF <10 EPI/LPF – Accept

Average no. of EPI/LPF >10 EPI/LPF – Reject

 

  1. Van Scoy has concluded that the sputum samples with more than 25 leukocytes per low power field should be accepted regardless of the number of epithelial cells present.

 

Average no. of WBC/LPF >25 WBC/LPF – Accept

 

  1. Geckler et al. has concluded that only samples with ≤25 epithelial cells per LPF should be accepted regardless of the number of leukocytes.

 

Average no. of EPI/LPF ≤25 EPI/LPF – Accept

Average no. of EPI/LPF >25 EPI/LPF- Reject

 

  1. Barry has described another method for judging the quality of expectorated sputum.

This scheme is similar to that of Bartlett which evaluates the ratio between white blood cells and squamous epithelial cells assigning + and – values. This minimizes the effect of variations in the thickness of material in different areas of the same slide.

 

Cells Score
if >150 WBC +3
if 76-150 WBC +2
if 1-75 WBC +1
if >25 EPI -3
if 16-25 EPI -2
if 5-15 EPI -1

 

Any positive score (sum of + and – values assigned) is taken as an acceptable sample, 0 or – value suggests that the sample should be rejected.

 

  1. Heineman and Radano have described another scheme for screening sputum specimens. They have recommended rejecting the specimens with more than 1 epithelial cell for every 10 white blood cells in average low power field is rejected.

 

Average ratio of WBC to EPI >10 WBC/EPI- Accept

Average ratio of WBC to EPI >10 WBC/EPI – Reject

 

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