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81 Annotations TB ALMOST WHILE YOU WAIT HITHERTO cultivation of tubercle bacilli has been too low a process to be of much use in diagnosis, but D. M. Pryce has described a micro -culture method which yields well-grown cultures within a week. Indeed, obvious signs of growth are visible with the TI’f in. objective in 24 hours, colonies may be just perceptible with the in. objective in 48 hours and in 3 days they are usually quite distinct. The method consists in drying a film of sputum on a glass surface, treating with 15% sulphuric acid, washing with water and incubating with blood hsemolysed with dis- tilled water or 1% saponin. After 7 days’ incubation the preparation is washed and stained with Ziehl- Neelsen and the colonies examined with the low power. The most typical colonies are long and stringy, later becoming twisted and coiled, but compact and bushy forms occur. Mycelial threads are found, and there is some suggestion that growth does not take place through- out the length of the thread but is restricted to certain points. Two methods of applying this dried-film culture technique are described. In one the sputum is spread on the floor of a petri dish. In the other one or more cir- cular patches of sputum are spread on a glass slide, dried, surrounded with a ring of bakelite, hard fibre or glass immersed in melted soft paraffin, and incubated in a moist chamber or with a coverslip to cover the ring. The method was tried out on sputa from 48 cases from the male wards of Harefield Sanatorium. In 28 growth was luxuriant and immediately obvious, and in 6 colonies grew which were sparsely distributed. The remaining 14 yielded no growth. The technique can also be applied to pus and caseous material. , PLASMA IRON ADVANCING biochemistry now allows the iron in blood plasma to be measured. When every precaution against haemolysis is taken there still remains a minute but fairly constant amount of iron measured in micro- grammes per c.cm. Vahlquist 2 gives the normal limits as 80-190 pg. per 100 c.cm. in men and 60-190 ug. in women with average figures of 122 and 104. In disease these levels differ ; but if we are to apply this newly acquired technique we must understand what has recently been learnt about the absorption and transport of iron. Hahn, Whipple and their co-workers 3 used " marked " radio-active iron to investigate this problem. They found, as Widdowson and McCance 4 had done, that iron absorption is proportional to iron needs and that once it is absorbed there is little excretion of iron. When iron salts are fed to dogs rendered ansemic by bleeding the radio-iron in the plasma rises rapidly, reaching a peak within 4-8 hours and then rapidly falling to resting level in 6-12 hours after feeding. Radio-iron appears in red blood-cells in traces as early as 4 hours after feeding and after 24 hours is present in considerable amounts. Only about 10% of the absorbed iron can be accounted for as haemoglobin iron, the rest presumably goes to iron stores. The plasma iron thus represents iron passing between the alimentary tract, the red-cell forming tissues and the iron stores, and we might expect that the response to a’test dose of iron would give some indication of the presence or absence of iron-deficiency. Vahlquist gives a series of " iron-tolerance curves " in various conditions. In a normal person the test dose causes a rise of serum iron to about 300 /kg. per 100 c.cm. in 4-6 hours, with return to normal level in 12 hours when fasting. The serum 1. J. Path. Bact. 1941, 53, 327. 2. Vahlquist, B. C. Acta pœdiatr., Stockh. suppl. 5, 1941. 3. Hahn, P. F., Whipple, G. D. et al. J. exper. Med. 1939, 69, 793; 70, 443. 4. Widdowson, E. M. and McCance, R. A. Biochem. J. 1937, 31, 2029. iron is below normal in anamia after haemorrhage, simple hypochromic anaemia of adults and children, and anaemia accompanying infection. In the first two a test dose causes a large rise in serum iron and after 12 hours the fasting level has not been regained ; in the infection anaemia there is little response. In pernicious anaemia the fasting level may be as high as 360 flg. per 100 c.cm. and a test dose of iron causes little further increase ; after liver treatment the resting serum iron falls to normal or below, and some iron deficiency is not un- common during the remission period. The fasting level is also high in hsemolytic anaemias. Waldenstrom 5 thinks that in clinical work serum-iron determination will be most useful in detecting iron deficiency. He has described patients with epithelial symptoms, such as sore mouth and dysphagia, but no anaemia, who have been shown to be deficient by the iron-tolerance test and who have responded satisfactorily to large doses of iron- so-called" latent sideropenia." The test is also useful in detecting iron-deficiency in patients with a normo- chromic anemia, in the remission phase of pernicious ansemia, and after gastric operations. There is one more finding that may prove useful : in acute hepatitis the serum iron may be raised as high as 370 p,g. per 100 c.cm., whereas in obstructive jaundice it is unaffected. Serum-iron determination then may take its place in the long list of biochemical tests for differentiating toxic and obstructive jaundice.. This sort of technique has also been used to estimate the value of the various iron preparations now used .. therapeutically. A test dose containing a standard quantity of iron is given and the change in serum iron followed. Moore and others,6 and Vahlquist, working on human subjects, found that ferrous salts were best absorbed, the rise in serum iron being unaffected by the absence of HCI in the stomach. Ferric salts, together with reducing agents like ascorbic acid or sodium formal- dehyde sulphoxylate, gave figures equal to ferrous iron ; ferric salts alone and other iron preparations were inferior. They noted that food influenced the absorption of iron, a subject discussed by Tompsett 7 who thinks that iron is only absorbed in the ferrous state. Hahn, Whipple and their colleagues,8 in their experi- ments with anaemic dogs on an iron-free but otherwise normal diet, got different results. They found the larger the doge of iron the smaller the percentage absorbed, so that it was best to give small divided doses. When conditions were optimal absorption was just as rapid whether the iron was given as a ferric or ferrous salt or even in an organic combination. The rate of utilisation of the absorbed iron was increased by previous iron feeding and by a diet of raw lean beef. By far the best results were obtained by intravenous injection of colloidal iron. How far these results on normal, temporarily anaemic dogs are applicable to abnormal anaemic men it is difficult to say. The use of intravenous iron suggests that a really efficient preparation to replace the unsatis- factory injectio ferri (BP) is needed. Enough has been said to show the usefulness of plasma or serum iron determinations. The methods available are, however, not simple, and for accuracy demand scrupulous cleanli- ness, iron-free reagents, and apparatus outside the usual range of laboratory equipment. The most favoured methods are based on the technique of Heilmeyer and Plbtner.9 A protein-free filtrate is prepared and the colour reaction between the iron in this filtrate and a substance like o-phenanthroline is used for the estimation. A photo-electric colorimeter or similar electrical instru- ment is necessary for accuracy. With these difficulties serum iron estimations are not likely to be popular, but 5. Waldenström, J. Nord. Med. 1941, 11, 2341. 6. Moore, C. V. et al. J. clin. Invest. 1939, 18, 553. 7. Tompsett, S. L. See Lancet, 1940, ii, 365. 8. J. exper. Med. 1940, 71, 731. 9. Heilmeyer, L. and Plötner, K. Das Serumeisen, Jena, 1937.
