S.K.Verma,MD1, R.Prasad,MD2
1. Professor, Department of Pulmonary Medicine
2. Prof & Head, Department of Pulmonary Medicine.

[emedpub – International Public Health : Vol 1:1] [Date of Publication: 06.30.2011]
ISSN pending

June 30, 2011 at 11:58 AM


Objectives:To determine if the results of PPD change by keeping PPD vials at room temperature (320 C-350 C) for 72-120 hours. The control comprised of PPD vials maintained in the cold chain (20C-80C).

Design of study: Prospective study from 15 July to 31 August, 2008.

Material and methods: For this study, both suspected and confirmed tuberculosis patients attending the Chest department during 15 July to 31 August, 2008 were recruited and were divided into different groups. For patients of first group (suspected tuberculosis patients), both vials of PPD were used and PPD was given in both the forearms of the same patient. Left forearm was given PPD which was refrigerated while on the right forearm, PPD kept at room temperature (320 C-350 C) for 72 – 120 hours was given. For the second group, (confirmed tuberculosis patients), patients were alternatively given PPD (one given refrigerated while the next patient was given PPD kept at room temperature).

Results: In the first group, of 80 patients, 72 (90%) had exactly identical results on both forearms. In the second group too, no difference was found with the size of PPD reaction.

Conclusions: The results suggest that one time loss of refrigeration up to 120 hours did not alter efficacy of PPD.


The tuberculin skin test was developed by Robert Koch in 1890, but the intradermal technique currently in use was described by Charles Mantoux in 1912. 1 The tuberculin most widely used is Purified Protein Derivative (PPD), which is derived from cultures of Mycobacterium tuberculosis. It is stated that maintenance of cold chain (20C-80C) is necessary for preservation of PPD. However, in a tropical country like India, where electricity supply is interrupted, the efficacy of PPD is frequently questioned. The authors therefore designed the study to find out whether the results of PPD changed by keeping PPD vials at room temperature (320 C-350 C) for 72-120 hours.

Patients and Methods:

In this experimental study, consecutive patients were assigned into two broad groups. In the first group, 118 patients of suspected tuberculosis who attended outpatient of the Department of Pulmonary Medicine at C.S.M.M.U, Lucknow from 15 July to 31 August, 2008 were enrolled in the study. In the second group, 45 sputum positive tuberculosis patients who were taking DOTS under RNTCP were enrolled.

For the patients of first group, both vials of PPD were used and a dose of 0.1 ml of 1TU PPD was given in both the forearms of the same patient. Left forearm was given PPD which was refrigerated (20 C-80 C) and in the right forearm, PPD given was kept at room temperature (320 C-350 C) for 72-120 hours.

For the second group, 45 patients with sputum smear positive were graded as: 15 patients with sputum smear status as 1 , 15 as sputum smear 2 , and 15 patients with sputum smear status 3 . Patients were given PPD, (one was given refrigerated and the next one given room temperature PPD). Skin tests were measure using a plastic scale at 72 hours after the intradermal injection.



Of 118 patients, 38 did not return for reading of PPD. For the remaining 80 patients, 45(56.25%) were males and 35(43.75%) were females. Of these 80 patients, 72 (90%) patients had exactly identical results on both forearms (Table 1).

In the remaining 8 patients (10%) with dissimilar results on both the arms, 6 patients were graded as non-reactive and one was reactive on both the arms (taking 10mm induration as cut-off line). In one patient, one arm had 3mm induration and in other it was borderline of 10mm. Though dissimilar, they did not, however, show significant difference in the final result of the patient (Table 2).

Table 1: Group 1 PPD results in both arms in patients with similar results (n=72).

Size of induration

Nil induration 24
<10mm induration 8
10-20mm induration 40

Table 2: Group 1 PPD results in both arms in patients with dissimilar results (n=8).

NO Right arm (PPD at room temperature)

Left arm (PPD at refrigerated temperature)
1. 4mm 0mm
2. 0mm 6mm
3. 3mm 10mm
4. 6mm 0mm
5. 0mm 7mm
6. 5mm 0mm
7. 4mm 0mm
8. 10mm 17mm


In this group, patients were graded further on the basis of sputum status into three sub-groups. Results obtained are shown in table 3.

As evident in table 3, even tuberculosis patients with comparable immune status and bacillary load had identical results with either of the two PPD vials.

Table 3: Group 2 PPD results in tuberculosis patients (n=45).



No.  of patients Amount of induration PPD at refrigerated temperature PPD at room temperature
1 15 >10mm 8 7
2 15 >10mm 7 8
3 15 >10mm 8 7


In the memorable year 1882, Robert Koch made his epoch-making discovery of the tubercle bacillus. In 1890, he made yet another discovery of tuberculin. Of the various skin tests devised to elicit hypersensitivity to tuberculosis infection, from the early subcutaneous injection to the latest intradermal injection with jet injector, the intradermal test first proposed by Charles Mantoux in 1908 is the most popular, so much so that tuberculin test is very often referred to as Mantoux test. PPD is prepared from heat concentrated old tuberculin by precipitating protein with trichloroacetic acid.2 To minimise its effect of adsorption Tween -80 is added to the diluent of PPD.3

The manufacturers of PPD provide the following information. “PPD should be stored between 20 and 80C. Do not freeze. Product which has been exposed to freezing should not be used. Tuberculin PPD solutions can be adversely affected by exposure to light. The product should be stored in the dark except when doses are actually being withdrawn from the vial. 4 A vial of Tuberculin PPD which has been entered and in use for 1 month should be discarded because oxidation and degradation may have reduced the potency.5 Do not use product beyond the expiry date.”

However, these recommendations are frequently ignored in the field. In a study done in Spain to determine if PPD RT-23 tuberculin from vials that had been open for a longer time could affect results of tuberculin tests and whether the solution could get contaminated. The study found that tuberculin did not appear to lose potency or sterility when vials had been used.6 A similar study was done in Makrana hospital, Makrana in 1995 where the author tried to investigate whether results of Mantoux test were affected by keeping the vials at room temperature for variable periods; that is at 24,48,72 and 96 hours and grouped patients separately. The study found no significant differences on comparing the results.7 In our test also we tried to check the effects of PPD at different temperature. Similarly, the results of this study suggest that one time loss of refrigeration up to 120 hours did not alter efficacy of PPD.

Limitations of the study:

This was a preliminary study. In order to validate its findings further, authors suggest that a larger study be undertaken to establish sensitivity of PPD to temperature variations over longer periods.  If found true in more studies, then the benefit of PPD can be extended even to remote parts of developing countries.


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  2. Bleiker JHA. The past, the present and the future of tuberculin test in tuberculosis control. Bull Int  Tuberc Lung Dis 1989,64:33-5
  3. American Thoracic Society. The tuberculin test. Official ATS statement. Am Rev Respir Dis 1984,124:356-65.
  4. Landi S, Held HR. Effect of light on tuberculin purified protein derivative solutions. Am Rev Respir Dis 1975;111: 52-61.
  5. Landi S. Effect of oxidation on the stability of tuberculin purified derivative (PPD) In: International symposium on tuberculins and BCG vaccine. Basel: International Association of Biological Standardization. Dev Biol Stand 1983; 58(B):545-52.
  6. Fernández VA, Gorís A, Otero M, Chouciño N, Vázquez R, Muñoz iñeiro MJ. Conservation of Purified Protein Derivative RT- 23 Tuberculin. Archivos de Bronconeumologia 2004:301-3.
  7. Ladha A. Effect of refrigeration on mantoux test result. Indian Paediatrics (letter)1995;32: 1036-7.
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