DIFFERENTIATION OF IRON DEFICIENCY FROM THALASSÆMIA TRAIT BY ROUTINE BLOOD-COUNT

449

Our experiments have demonstrated that the capacity of sera from patients

with

hypocomplementasmic

M.c.G.N.

to

break down pure C3 in normal human serum is due to activation of the alternative pathway which does not require any of the normal-pathway components. The relation of this phenomenon (and the distinctive abnormalities of the complement system which may be presumed to result from it) to the pathogenesis of glomerulonephritis remains uncertain. We thank Dr J. S. Cameron for supplying the sera, Miss J. Fallows and Mr N. Amos for skilled technical assistance, and Dr M. Hobart for preparation of C3. The Wellcome Trust provided

patients’

support. Requests for reprints should be addressed

generous

to

D. K. P.

certain that at least at some stage in the natural history of the disease, C3 catabolism is accelerated; two groups 10,11 have reported accelerated C3 turnover in some hypocomplementxmic patients with M.C.G.N. It is interesting to speculate whether accelerated C3 turnover might have resulted from inadvertent administration of C3b in the labelled C3 preparations. The mode of action of the alternative-pathway factors is not fully understood, but that they are necessary for C3 breakdown by nephritic C3-convertase is evident by the failure of C3NeF fraction to break down C3 in the C3 preparation containing C3b, even when G.B.G. was added to the reaction mixture. A

hypothesis which takes into account our present knowledge of the system is presented in an accompanying paper. 12 A provisional scheme (based on this hypothesis) showing how C3NeF may generate a C3convertase is outlined in the accompanying figure. In this scheme nephritic C3-convertase is, in fact, identical with the alternative pathway or bypass convertase-i.e., it is the activated form of G.B.G. Our conclusion that C3b is required for the generation of a nephritic C3-convertase would then be extrapolated to a general requirement for C3b in the generation of an alternative pathway or bypass convertase. Previous work 13-15 has shown that C3 or a C3 breakdown product is a requirement for alternative-pathway activation in vitro. Our results indicate that C3b is the form in which C3 is required for the in-vivo reaction. Recognition of the capacity of C3b to activate the alternative pathway provides an explanation for the finding of low levels of G.B.G.9m1 in diseases such as systemic lupus erythematosus, where there is unequivocal evidence of complement activation via the normal pathway. In such conditions C3 activation would primarily result from the action of a normal convertase (i.e., C42) but C3b generated by this reaction would then activate the alternative pathway. It is also possible that in some circumstances activation of the alternative pathway might continue after the primary stimulus to the normal pathway had ceased. Such a sequence would explain the events in acute post-streptococcal nephritis where circulating C4 levels are only transiently reduced but reduction in C3 is

more

protracted. 16

REFERENCES 1. 2. 3.

4. 5. 6. 7. 8.

9. 10.

11.

12. 13. 14. 15. 16.

West, C. D., McAdams, A. J., McConville, J. M., Davis, N. C., Holland, N. H. J. Pediat. 1965, 67, 1089. Gotoff, S. P., Fellers, F. X., Vawter, G. F., Janeway, C. A., Rosen, F. S. New Engl. J. Med. 1965, 273, 524. Cameron, J. S., Glasgow, E. F., Ogg, C. S., White, R. H. R. Br. med. J. 1970, iv, 7. Spitzer, R. E., Vallota, E. H., Forristal, J., Sudora, E., Stitzel, A., Davis, N. C., West, C. D. Science, 1969, 164, 436. Vallota, E. H., Forristal, J., Spitzer, R. E., Davis, N. C., West, C. D. J. exp. Med. 1970, 131, 1306. Peters, D. K., Martin, A., Weinstein, A., Barratt, T. M., Cameron, J. S., Ogg, C. S., Lachmann, P. J. Clin. exp. Immun. 1972, 11, 311. Lachmann, P. J. Immunochemistry, 1971, 8, 81. Lachmann, P. J., Hobart, M. J., Aston, W. P. Handbook of Experimental Immunology (edited by D. M. Weir) (in the press). Williams, D. G., Charlesworth, J. A., Evans, D. J., Peters, D. K., Cameron, J. S., Morel-Maroger, L., Kourilsky, O. Unpublished. Herdman, R. C., Pickering, R. J., Michael, A. F., Vernier, R. S., Fish, A. J., Gewurz, H., Good, R. A. Medicine, Baltimore, 1970, 49, 207. Hunsicker, L. G., Ruddy, S., Carpenter, C. B., Schur, P. H., Merrill, J. P., Müller-Eberhard, H. J., Austen, K. F. New Engl. J. Med. 1972, 287, 835. Lachmann, P. J., Nicol, P. Lancet, 1973, i, 465. Müller-Eberhard, H. J., Gotze, O. J. exp. Med. 1972, 135, 1003. Alper, C. A., Colten, H. R., Rosen, F. S., Rabson, A. R., MacNab, G. M., Gear, J. S. S. Lancet, 1972, ii, 1179. Nicol, P. A. E., Lachmann, P. J. Immunology (in the press). Kohler, P. F., Ten Bensel, R. Clin. exp. Immun. 1969, 4, 191.

DIFFERENTIATION OF IRON DEFICIENCY FROM THALASSÆMIA TRAIT BY ROUTINE BLOOD-COUNT

J. M. ENGLAND

PATRICIA M. FRASER

Department of Hœmatology, and Division of and Statistics, Clinical Research Centre, Harrow, Middlesex

Computing

Summary

Some

cases

heterozygous

of iron

deficiency and &bgr;-thalassæmia (thalas-

sæmia trait) can be differentiated on the basis of the red-blood-cell count (R.B.C.) alone. However, in 72 patients presenting with microcytosis, a simple discriminant function derived from the mean cell volume (M.C.V. in fl.), the R.B.C. 106 per µl., and the hæmoin g. per 100 ml.) correctly of the cases studied (99%). The function took the following form: D.F.’=M.C.V.-R.B.C. -(5×Hb)-3·4. A positive value indicated iron

globin concentration (Hb identified all but

one

450 and a negative value indicated thalassæmia trait. This function is not applicable in pregnancy.

deficiency

Introduction

IT is often impossible to differentiate between iron deficiency and heterozygous -thalassaemia (thalassaemia trait) either by examining the red-blood-cell morphology or the red-blood-cell indices. In these circumstances it is customary to confirm the suspected diagnosis by measuring the serum iron and ironbinding capacity and the level of haemoglobin A2.1 The introduction of electronic cell counters has meant that reliable values for the mean cell volume (M.c.v.) and red-blood-cell count (R.B.C.) can now be obtained routinely. It therefore became appropriate to see whether this information alone was sufficient for the routine differentiation of iron deficiency from thalassxmia trait. Materials and Methods

Haemoglobin, R.B.c., and M.c.v. were measured on the model S Coulter Counter. The hxmoglobin reading was set by comparison with blood-samples read against cyanmethxmoglobin standards. The R.B.C. was standardised using a model FN Coulter Counter. The M.c.v. was standardised indirectly by setting the machine to read the packed-cell volume (P.c.v.) 1-5% lower than the value obtained in the microhxmatocrit. This allows for plasma trapping in the microhaemaiocrit and standardises the M.c.v. on the counter, since on this machine the M.c.v. and P.c.v. are related by the formula:

When the machine is adjusted in this manner the normal range for M.c.v. becomes 80-90 fl. in young hospital staff.2 Haemoglobin A2 and F were determined by the method described by Dacie and Lewis.3 Serum iron and ironbinding capacity were measured as described by Young and Hicks.’

Fig. 1-Distribution of R.B.C., Hb, and M.C.V. iron deficiency and thatassaemia trait.

Results

The iron-deficient group of 25 patients had a mean serum-iron of 4-8 mole per litre (range 1-5-9-4 jmole per litre). Their iron-binding capacity was correspondingly increased (mean 81-6 µmole per litre, range 75 097-5 pinole per litre). The 28 patients with thalassxmia trait all had an increased percentage of HbA (range 3-1-6-7%, mean 4-3%). All the patients had small red blood-cells with an M.c.v. of less than 80 fl. In fig. 1 the R.B.c., Hb, and M.c.v. have been plotted for the members of the two groups. For all three indices the overlap is considerable, so that it is not

Patients with iron deficiency were selected from the clinical chemistry department records if they had a serumiron below 12 mole per litre and an iron-binding capacity of 75 mole per litre or above. Patients with thalassxmia trait were selected from the hxmatology department records if they had over 3°o of HbA2 and no other abnormal haemoglobin on starch-gel electrophoresis (’ Tris ’ buffer, pH 8-9). After the patients had been selected for study their blood-counts were obtained from the hsematology department records.

RED-BLOOD-CELL INDICES IN PATIENTS WITH IRON-DEFICIENCY ANEMIA AND THALASSAMIA TRAIT

Mean - 1-96

patients with

25 patients with iron deficiency and 28 patients with thalassxmia trait were studied in the first instance and a further 19 new patients were studied after derivation of a discriminant function (D.F.).

Patients

*

in

x s.D.

t t test.

451

possible to separate the groups completely on the basis of any single index. The R.B.c. gives the best separation, but even so it can be calculated that nearly 17 °o of the members of each group will have values which fall in the area of overlap. The ranges covered by the three calculated indices (p.c.v., mean corpuscular haemoglobin [M.c.H.], and mean corpuscular haemoglobin concentration [M.C.H.C.]) overlapped even more. A series of normal plots5 showed that all the indices in both groups were approximately normally distributed. All the indices show a significant difference between the means of the two groups (see accompanying table). However, although the groups taken as a whole are significantly different, the considerable overlap between the individual patients is reflected in the ranges covered by 95 % of the members of each group-i.e., mean ± 1 -96 x s.D. A very pronounced improvement in the ability of the red-blood-cell indices to discriminate between the microcytic anxmia of iron deficiency and that of thalassxmia trait was obtained by combining the indices in a D.F.6A weighted linear combination of the three measured indices (R.B.c., Hb, M.c.v.) separated the two groups almost completely. The D.F. took the following form: D.F. =(0-23 x M.c.v.) -(0-22 x R.B.c.) — (0-93 x Hb) -3-00. In fig. 2 the values

Fit. 2-Distribution of M.C.V.

a

of the D.F. for each

been plotted and all the patients with iron deficiency have a positive value and all but one of those with thalassaemia trait have a negative value. Also shown on fig. 2 are the ranges in which 95 % of the patients within each group might be expected to fall-i.e., mean ± 1 -96 x s.D. At this level there is a small overlap between the ranges, so that the 5-4% of values from each group expected to fall into this area should be interpreted with caution. The discriminant analysis was repeated using other combinations of the indices and all six indices, but the combination of the three measured indices was most effective in discriminating between the two groups of patients. The values of the D.F.S were calculated for a further 9 new patients with iron-deficiency anaemia and 10 new patients with thalassaemia trait. Their values are also plotted on fig. 2 and correctly show positive and

negative discriminants, respectively. An attempt

was

then made

to see

whether the

D.F.

simplified without loss of precision. Simple rounding, multiplication, and subtraction of a constant could be

enabled the function

to

be modified

as

follows: D.F.’=

M.C.V.-R.B.C.-(5 xHb)-3’4. The value of this simplified function was calculated for the 53 original

patient have

D.F. derived from the R.B.C.,

Hb, and

Fig. 3-Distribution of a simplified D.F. (D.F.’) derived from the R.B.C., Hb, and M.C.V.

452

patients and the 19 new patients. Again all the patients with iron-deficiency ansemia had a positive discrimi-

TREATMENT OF DRUG OVERDOSAGE WITH NALOXONE, A SPECIFIC

and all but one of those with thalasssemia trait a negative value (fig. 3). However, the overlap between the ranges is slightly increased by the approximation, so that 6-60,, of patients from each group would be expected to fall into the area of uncertainty. nant

NARCOTIC ANTAGONIST

L. E. J. EVANS C. P. SWAINSON

Regional Poisoning Treatment Centre, Royal Infirmary, Edinburgh

Discussion

possible discriminate between iron deficiency and thalassaemia trait on the basis of the M.C.V., R.B.C., and Hb. A simplified D.F. (D.F.’) has been developed for use with a model S Coulter Counter which is adjusted to read a true P.c.v. The D.F.’ should only be used when thalassxmia trait or iron deficiency are suspected in individuals with microcytosis, and it is pointless to calculate the D.F.’ for individuals with a normal M.c.v. -i.e., > 80 fl. Other D.F.s would need to be calculated if the counter was adjusted in some other way, or if another machine was used. This method may find a place both in routine laboratory use and in population screening as advocated by Modell et al.7 The D.F.’ is only of value in uncomplicated cases of iron deficiency or thalassxmia trait. Hsemodilution after haemorrhage or in pregnancy has given misleading results, and we have noticed that this may also occur when subjects with thalassxmia trait become anxmic with malaria. Mild iron deficiency probably does not interfere with the discrimination of thalassxmia trait, because several of the cases of thalasssemia trait would undoubtedly have mild iron deficiency. The one patient with thalassarmia trait and a positive discriminant may also have been iron deficient, but a further blood-sample could not be obtained to test this hypothesis. Severe iron deficiency and coexistent thalassxmia trait was only seen in one patient, who had chronic gastrointestinal bleeding. She presented with a severe microcytic ansemia (Hb 6-2 g. per 100 ml., R.B.c. 4-04 x 106 per 1., M.c.v. 51 fl.) and the serum-iron was 3-4 ,mole per litre, with an iron-binding capacity of 84-9 jjunole per litre. The simplified discriminant function (D.F.’) was calculated to be 12-6, indicating iron deficiency. After iron therapy the hxmoglobin rose to 15-4 g. per 100 ml., with 6-71 x 106 red blood-cells per 1., but the M.c.v. remained low at 71 fl. The D.F.’ was then recalculated and found to be -16-1, indicating that thalassxmia trait was present. This diagnosis was confirmed when the HbA2 was found to be 4-7%, with an HbF of 0-800. It appears, therefore, that when severe iron deficiency and thalassaemia trait coexist, the simplified D.F. (D.F.’) indicates iron deficiency. However, in these circumstances measurement of the HbA2 levels may be of little value because normal levels may be found.The only way to assess this situation is to correct the iron deficiency and then re-evaluate the It is

to

P. ROSCOE L. F. PRESCOTT

Naloxone (N-allylnoroxymorphone) is potent narcotic antagonist which is devoid of agonist activity. Nine patients with narcotic analgesic overdosage recovered consciousness immediately after intravenous injection of 0·4-1·2 mg. of naloxone given in divided doses over 3 minutes. There was a striking increase in respiratory rate and volume accompanied by a rise in systolic bloodIn contrast, pressure and dilatation of the pupils. naloxone did not produce any change in the level of

Summarya

consciousness, respiration, blood-pressure, pulse-rate,

pupil size in thirteen deeply unconscious patients poisoned with a variety of non-narcotic centralUnlike nalorphine, nervous-system depressants. naloxone readily reverses the effects of pentazocine and has no intrinsic respiratory depressant activity. or

Introduction

ACCIDENTAL and deliberate overdosage with narcotic analgesics is an increasingly common problem, and is especially dangerous because of severe respiratory depression. Nalorphine and levallorphan are narcotic antagonists which are used routinely as specific antidotes for the treatment of narcotic overdosage. However, these drugs are not ideal antagonists, since they are also partial agonists and can themselves cause serious respiratory depression. The intravenous injection of nalorphine has been advocated as a diagnostic test in drug-induced coma, but this can be hazardous if narcotic analgesics have not been taken. In such circumstances further depression of respiration may occur, especially in patients who have taken

barbiturates. Naloxone (N-allylnoroxymorphone,Narcan ’) is a narcotic antagonist which is virtually devoid of agonist activity. It has no intrinsic respiratory or cardiovascular depressant actions, 2,3and, unlike nalorphine, it can reverse the effects of pentazocine. Naloxone can reverse the mild respiratory depression caused by therapeutic doses of narcotic analgesics, including morphine, pethidine, pentazocine, and oxymorphone.1, 2,4-7 However, apart from a single report of its use in methadone poisoning8 there is no detailed

1.

patient.

2.

We thank Mr M. J. R. Healy and Dr 1. Chanarin for their helpful comments; the members of the departments of hxmatology and clinical chemistry who carried out the analyses; and the members of the division of computing and statistics who assisted with the data-processing. J. -M. E. is supported by an M.R.C. fellowship. Requests for reprints should be addressed to P. M. F.

3.

DR ENGLAND, DR FRASER: REFERENCES Weatherall, D. J., Clegg, J. B. The Thalassæmia Syndromes. p. 113. Oxford, 1972. England, J. M., Walford, D. M., Waters, D. A. W. Bv. J. Hæmat 1972, 23, 247. Dacie, J. V., Lewis, S. M. Practical Hæmatology, p. 514 London.

1968. 4. 5. 6.

Young, D. S., Hicks, J. M. J. clin. Path. 1965, 18, 98. Healy, M. J. R. Br. med. Bull. 1968, 24, 210. Healy, M. J. R. in Mathematics and Computer Science in Biology and Medicine, pp. 93-102. H.M. Stationery Office, London, 1965 7. Modell, C. B., Benson, A., Payling Wright, C. R. Br med. J 1972 iii, 737. 8. Wasi, P., Disthasongchan, P., Na-Nakorn, S. J. Lab du. Mec 1968, 71, 85.