616. 155. 1-76. 5 : 616-001 , 17

MORPHOLOGICAL CHANGES IN THE RED CELLS IN R,ELATION TO SEVERE BURNS

ALEXANDER BROWN From the Royal Infirmary, Glasgow

IN a study of the hsmatological changes in burns (Brown, 1944), it was shown that burns involving more than 15 per cent. of the body surface are frequently followed by a rapidly occurring moderately severe ansmia, the development of which is associated with a slight and variable increase in the mean corpuscular volume, increased erythrocyte fragility and increased plasma bilirubin and urobilin excretion. I n the original publication only a preliminary report was made on the changes in size of the red cells. The results of further study of the material and findings obtained in the original investigation are now presented. These studies are concerned with the variations in mean corpuscular volume (M.C.V.), mean corpuscular diameter (M.C.D.) and mean corpuscular average thickness (M.C.A.T.), in relation to variations in osmotic fragility and the mechanism of production of the anaemia.

Methods

The three patients referred to here and the methods of estimation of hsmoglobin and of saline fragility have been described in the original report, to which reference may be made for full details.

Hsmoglobin was estimated photo-electrically, using carboxyhsmoglobin as the pigment. Saline fragility was estimated quantitatively, percentage lysis in each concentration of saline being obtained by comparison with a standard equivalent to 50 per cent. lysis, using a Duboscq colorimeter.

Red cell diameters were measured by microprojection on to millimetre-ruled graph paper at a magnification of 1000 diameters. Each red cell diameter recorded was the average of two measurements at right angles to each other. Evenly spread coverslip preparations stained by Leishman’s stain were used and 400 cells were measured in each film.

Results

I n the original study, blood films were prepared from all samples of blood obtained for haematological investigation. No significant alteration in the size or shape of the erythrocytes was apparent in patients mildly burned. I n cases with severe burns it was evident, oven from cursory inspection of stained films, that microspherocytosis was a prominent feature within a few hours of burning. It persisted

367

368 A. BROWN

14.5 37.0

94 '

6.52

in gradually diminishing degree for 2-4 days after injury. Fragmenta- tion of red cells was observed only in films made within a few hours of burning. I n such cases small irregular portions of red cells apparently fully hzmoglobinated and measuring 0.5-2.0 p in diameter were numerous. I n some instances a few ghost cells and non-haemo- globinated fragments were also seen. I n preparing the distribution curves illustrating changes in red cell diameter, fragmented cells were ignored as far as possible. These curves are not shown here. but the mean values for cell diameter are contained in the tables.

... 35.0

104 6.78

Case reports

Case 1 (no. 46 in original series). Fernale aged 10, 80 per cent. of body surface This child sustained extensive third degree burns about 1 hour before

The findings in the 4-day survival period are recorded burned. admission to hospital. in table I.

TABLE I Blood jindings in case 1

Time after burning (hours)

Hb. (g. per 100 c.c.) . P.C.V. (per cent.) 46.0 M.C.V. ( ~ p ) . : 1 104 M.C.D. (p) . . 1 6-23 M.C.A.T. (p) . . , 3.40 M.C.F. (per 0.436

cent* ! NsC1) Residuh lysis (per i 15.0

cent.) * 100 C.C.)

Plasma t Hb (9. per

3.5

12.1 32.0

94 6.56 2.82 0.440

10.0

0.5

5 1 8

10.0 1 8.0

o'6 ~ 0.4

18

12.4 30.0

83 7.15 2.06 0.400

5.0

:0.1

54

14.0 37.5

108 7.75 2.30 0.373

3 .O

0

i 2

11.2 32.5

109 7.85 2.25 0.328

0

0

96

10.5 30-6

105 7.75 2,23 0.324

0

0

* In this case lysis which persisted in concentrations of NaCl above 0.50 per cent. t Hsmoglobinaemia and haemoglobinuria were present in this case.

On admission there was evidence of hsmoconcentration. The 3Z.C.V. was slightly above normal, although the average diameter was diminished. An increase in the M.C.A.T. was associated with a slightly raised median corpusculrtr fragility (M.C.F.) and with haemolysis amounting to 15 per cent. in all con- centrations of sodium chloride above 0.50 per cent. Haemoglobinzemia and haemoglobinuria were prominent features. During the period of survival the M.C.D. progressively incremed, the M.C.A.T. and the osmotic fragility diminished, and the hamoglobinaemia and haemoglobinuria disappeared. Frag- mented cells were numerous in the first two samples but rapidly disappeared thereafter. Microspherocytes were obvious in early films and, even in the last sample, 96 hours after injury, a few very deeply stained cells still remained.

Case 2 (no. 48 in original series). Female aged 7, 40 per cent. of body surface burned. This child was admitted to hospital with severe third degree burns about 3 hours after injury and the first samples of blood were obtained almost immediately (table 11).

The M.C.V. was subnormal and this was associated with a reduction in diameter rather than in thickness. The M.C.F. was within normal limits and there was neither

She died three weeks later of agranulocytosis. On admission there wm evidence of haemoconcentration.

RED CELL CHANGES I N SEVERE BURNS 369

16 36 1 60 I------

visible haemoglobinaemia nor haemoglobinuria. At the 6th hour the spherocytosis had increased and the M.C.F. was found to be abnormally high. Thereafter osmotic fragdity returned to normal and to subnormal, in association with an increase in the M.C.D. and a reduction in the M.C.A.T.

TABLE I1

Blood findings in case 2

72 120

Time after burning (hours)

' 3 1 6 1 6 ' 26 1 4 0 1 6 5 I 9 6 1 1 2 0

20.4 ~ 13.2 117.5 ' 16.9 ~ 14.0 52.5 31.5 46.0 ' 4 1 . 5 40.0

~ 94.5 ~ 91.0 1 102 181.0 95.5

Hb. (g. per 100 C.C. P.C.V. (per cent.) M.C.V. ( c . , ~ ) .

12.4 34.5

103

M.C.D. (pj ' . M.C.A.T. (p) . M.C.F. (per cent. NaCl)

18.7 50.0 75.0 6.75 2.10 0.424

12.6 10.7 35.0 28.5 86.0 80.0 6.27 6.89 2.80 2.14 0.460 0.394

12.2 33.0 98.0

7.50 2.22 0.404

12.0 38.0 93.5 7.63 2.04 0.385

9.5 24.5 87.0

7.75 1.85 0.352

In this case fragmentation of the red cells was not an obvious feature, and microspherocytosis was less obvious than in case 1. The change was, however, still visible in films 96 hours after burning.

Male aged 35, 40 per cent. of body surface burned. The haematological findings are shown in table 111. This patient survived, but was left with great disability owing to destruction of tissue in the limbs.

Case 3 (no. 58 in original series). Almost the entire burn in this case resulted in whole skin loss.

TABLE 111 Blood findings in case 3

Hb. (g. per 100 c.c.) . P.C.V. (per cent.) . M.C.V. (0.p) M.C.D. (p ) . M.C.A.T. (p ) . M.C.F. (per cent. NaCl)

I-- 119.0 119.1

50.0 50.5 98.0 98.0 6.77 6.67 1 7.15 6.92 7-06 I 7.24 7.49 7.66 2.73 1 2.82 2.35 ~ 2.41 ~ 2.60 j 1.97 1 1.95 ~ 2.12 0.476 I 0 .448 ' 0.454 0.444 0.406 0.380 0.360 0.340

! I 1

Three hours after injury haemoconcentration was a marked feature. The M.C.V. was normal but the values for diameter and thickness indicated a significant degree of spherocytosis. The increase in M.C.A.T. was maintained throughout the first 36 hours and in the first 4 samples of blood (to 16 hours) there was a great increase in osmotic fragility. The M.C.F. returned to normal and subnormal as the cell diameter increased and the M.C.A.T. fell to about 2 p.

In this as in the previous case fragmentation was not an obvious feature even in the first specimen of blood. Microspherocytosis was less marked than in the first case (case 46), and it was no longer seen after 36 hours.

Discussion The three patients described in this report are examples of very

severe burns. All developed anaemia after the injury, and in one (case 1) hsmoglobinaemia and haemoglobinuria were prominent features.

370 A . BROWN

Investigation of a large series of burns of different degrees of severity (Brown, 1944) has shown that anaemia of a hamolytic type tends to follow those involving more than 15 per cent. of the body surface. A summary of the changes in M.C.V. was presented in the original report. These were so variable as to provide inconclusive evidence of definite alterations in cell size due to the injury. The reason is clear. The mean corpuscular volume is a function of diameter and thickness : it may be little altered if spherocytosis is produced at the expense of diameter. Such a change has been shown to occur after severe burns. The most severe injuries are associated not only with microspherocytosis but also with actual fragmentation of the red cells, intravascular hemolysis and hamoglobinuria. Less severe damage is associated with microspherocytosis alone, which is obvious in stained films and is further revealed by the rise in the M.C.A.T. It is associated with a demonstrable increase in osmotic fragility .

It has long been known that morphological changes occur in the blood of a burned patient, yet little information is to be obtained from modern works on hematology as to the nature of these changes and their etiology. Whitby and Britton (1944) state that " the mechanism of hsemolysis in severe burns has not been fully elucidated, but it is supposed to be due either to secondary infection, or more probably to absorption of hamolytic protein cleavage products ". Wintrobe (1942) limits himself to the statement that " Severe hemo- lytic anemia with hemoglobinuria is said to occur . . . sometimes following extensive burns ".

I n reviewing the literature on the effect of heat on the blood and of severe burns on the patient, no reference has been found indicating that hsemolytic protein cleavage products may be responsible for the production of haemolytic anaemia after such injuries. On the other hand, there is good evidence that heat is responsible for certain morphological changes in the red cells whereby these are rendered unduly susceptible to the physiological trauma of the circulation and intravascular hamolysis occurs.

The earliest studies of changes in the red cells in burns were undertaken in an attempt to explain death from burns shock. Schultze ( 1865) observed that crenation and fragmentation followed exposure of the blood to temperatures of 51-52°C. Similar findings were recorded by von Lesser (1880), Silbermann (1890), Burkhardt (1904-05) and Helsted (1906). Experimental burns in animals were found to be followed by similar changes in the blood within a short time of injury (von Lesser, 1880 ; Markusfeld and Steinhaus, 1895 ; Pfeiffer, 1905). Hsemoglobinaemia and hemoglobinuria were observed in animals burned by scalding and in animals transfused with blood from a burned animal. Isaacs et al. (1924-25) confirmed the morpho- logical changes described in heated blood, and observed that red cells heated above 50" C. showed increased osmotic fragility. Locke (1902)

RED CELL CHANGES I N SEVERE BURNS 371

recorded fragmentation of red cells in two fatal cases of burns within 1$ hours of injury.

These experimental and clinical findings have recently been confirmed by Shen and Ham (1943), who demonstrated the occurrence of intravascular haemolysis with haemoglobinaemia and haemoglobinuria in several cases of severe burns. I n some cases the red cells showed increased osmotic fragility which was associated with microsphero- cytosis as estimated by inspection of stained films. It has previously been shown (Brown) that burned patients, roughly in proportion to the severity of the burn, are liable to become anaemic within a week of the injury and that the development of the anaemia is associated with a slight and variable increase in the M.C.V. and increased osmotic fragility of the red cells. Plasma bilirubin and urinary excretion of urobilin are increased (Anderson and Semeonoff, 1944). The greatest change in fragility was found to occur in association with haemoglobinaemia and haemoglobinuria in patients with extensive and deep burns. In the present investigation it has been shown that these alterations are accompanied by a demonstrable increase in the mean corpuscular average thickness of the red cells. The impression of microspherocytosis obtained from inspection of stained films (Shen and Ham) has thus been confirmed.

The fundamental cause of the change in the red cells leading to hsmolysis is probably the direct action of heat. When blood is heated rapidly to 51-55" C. and then immediately cooled to 37" C., the changes produced are exactly those which have been shown to occur in the burned patient. In addition, these changes are independent of the fluid in which the cells are suspended (Shen and Ham). These authors have also shown t'hat blood which has undergone such changes is abnormally susceptible to mechanical trauma artificially produced. The probable mechanism of haemolysis in burns thus becomes increasingly evident.

Conclusions

Investigation of the morphological changes occurring in the red cells of three very severely burned patients has shown that fragmenta- tion of the red cells and microspherocytosis occur within a few hours of the injury. Microspherocytosis was seen in stained films and was shown to be associated with an increase in the mean corpuscular average thickness. Fragmentation of the red cells was most obvious in the most severely burned patient and was associated with haemo- globinaemia and haemoglobinuria.

The morphological changes occurring in the red cells in burns can be attributed to the direct action of heat on the cells. The maximum effect is immediate fragmentation and destruction. If the damage is less severe microspherocytosis is produced and the affected cells are unduly susceptible to the physiological trauma of the circulation.

372 A. BROWN

Depending on the magnitude and rate of haemolysis, haemoglobinaeinia and haemoglobinuria may occur.

'The author was in receipt of a part-time grant from the Medical Research Council during the original investigation (1942-43). He desires to acknowledge his indebtedness to Professor J. W. S. Blacklock for permission to conduct the investigation in the Department of Pathology at the Royal Infirmary, Glasgow, during this period. Thanks are also due to Professor L. J. Davis for helpful criticism.

REFERENCES

ANDERSON, A. B., AND SEMEON- OFF, E.

BROWN, A. . . . . . . . BURKHARDT . . . . . . . HELSTED, A. . . . . . . ISAACS, R., BROCK, u., AND MINOT,

VON LESSER, L. . . . . . . LOCKE, E. A. . . . . . .

G. R.

MARKUSFELD, ST, AND STEIN-

PFEIFFER, H. . . . . . . SCHULTZE, M. . . . . . . SHEN, S. C., AND HAM, T. H. . . SILBERMANN, 0. . . . . . WHITBY, L. E. H., AND BRITTON,

WINTROBE, M. M. . . . . .

HAUS, J.

C. J. C.

1944. Medical Research Council, Spec. Rep. Ser. no. 249, London, pt. V, p. 166.

1944. 1904-05. Arch. Llin. Chir., Ixxv, 845. 1906. Ibid., lxxix, 414. 1924-25. J . Clin. Invcst., i, 425.

Ibid., pt. IV, p. 114.

1880. Arch. path. Anat., Ixxix, 248. 1902. Boston Med. and Surg. J., cxlvii,

1895. 480.

Cbl. allg. Path., vi, 1.

1905. Arch. path. Anat., clxxx, 367. 1865. Arch. rnicr. Anat., i, 1. 1943. New England J . Med., ccxxix, 701. 1890. Arch. path. Anat.,. cxix, 488. 1944. Disorders of the blood, 4th ed.

(revised reprint), London, p. 257. 1942. Clinical hematology, London, p. 378

or 427.*

* There are separate printings of this book, differently paginated.-ED.