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International Journal of Gynecology SC Obstetrics 52 (1996) 33-36 ... 0020-7292/96/$15.00 0 1996 International Federation of Gynecology and Obstetrics.
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GYNECOIANY & OBSTETRICS International Journal of Gynecology SCObstetrics 52 (1996) 33-36

Article

Screening for anemia in pregnancy with copper sulfate densitometry L.R. Pistorius*, Department

of Obsretrics

M. Funk, R.C. Pattinson, G.R. Howarth

and Gynecology,

Kalafong

Hospital

and University

of Pretoria,

Pretoria,

South Africa

Received 17 May 1995; revision received 8 August 1995; accepted 15 August I995

Abstract Objective: The copper sulfate method of screening for anemia was evaluated to determine its accuracy in antenatal patients. Method: In an antenatal clinic in a tertiary referral center, which also serves a local urban black community, 100 antenatal patients were prospectively tested for anemia by Coulter hemoglobin analysis in comparison with the copper sulfate test. The respective accuracy and costs of the tests were evaluated. Results: Once initial technical difftculties had been overcome, the copper sulfate test proved accurate in detecting a hemoglobin level < 10 g% in pregnancy (sensitivity 94%, specificity 95%, positive predictive value 80%, negative predictive value 99%). The cost of the copper sulfate test is estimated to be less than 0.3% that of the Coulter test. Conclusion: The copper sulfate test is accurate and inexpensive, and can be recommended for screening for anemia in pregnancy. Keywords: Pregnancy; Anemia;

Screening; Copper sulfate

1. Introduction In pregnancy the hemoglobin concentration is lowered as an effect of a disproportionate increase in plasma volume relative to the increase in red cell mass. This apparent anemia is a normal physiologic phenomenon and is probably beneficial to the mother and fetus [l]. However, true anemia in pregnancy is associated with an increased perinatal mortality and might be a direct or indirect contributor to a significant proportion of maternal deaths in the developing world [2]. l

Corresponding author, Fax: +27 12 3739031

Screening for anemia in pregnancy is therefore considered an essential part of antenatal care [2]. As the clinical diagnosis of anemia is notoriously inaccurate [3,4], different objective methods are in use to screen for it. These include using a portable hemoglobinometer such as the BMS hemoglobinometer (Premed, Pretoria, South Africa, microhematocrit estimation, and even a Coulter count. Whereas these methods are accurate [5,6], they are expensive and require a power source as well as technical support in case of instrument malfunction. In the search for technology appropriate to the developing world, consideration should be given to the copper sulfate

0020-7292/96/$15.00 0 1996 International Federation of Gynecology and Obstetrics SSDI 0020-7292(95)02559-U

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method of screening for anemia. This method has been tested in a small Jamaican study of pregnant women, with good results, although the investigators did not specify the specific gravity (SG) of the copper sulfate solution used, the prevalence of anemia in their population, or their sample size 171.

The copper sulfate method of screening is in current use for screening potential blood donors. It was originally developed during World War II, and subsequently refined [8,9]. According to Archimedes’ principle, an object will float in a solution of higher density (or SG), or sink in a solution of lower density. When a drop of blood is immersed into a solution of copper sulfate, a sac of copper proteinate forms on the surface of the drop, preventing a change in its SG for lo-15 s. When a drop of blood falls into a solution of copper sulfate, its momentum will carry it l-2 cm below the surface. Within 5 s, the momentum of the fall is lost, and the drop would then rise (if its SG is lower than that of the copper sulfate solution), continue to sink (if its SG is higher than that of the solution), or remain stationary for a few seconds and then sink (if its SG equals that of the solution) [8]. The SG of the drop of blood would depend on the SG of the plasma and the blood cells. For a constant plasma SG, the hemoglobin value could then be deduced from a nomogram [9]. Conversely a copper sulfate solution of specific SG could be used for screening for the corresponding hemoglobin value. The advantage of the copper sulfate solution test is that it requires no power source, is stable (provided that evaporation of the stock solution is avoided), and accurate from 4 to 40°C [8]. It would seem to be the ideal test to screen for anemia in pregnancy. The following study was devised to test this hypothesis. 1.1. Study objectives

The objectives of the study were to: (1) determine the correct SG of copper sulfate aqueous solution to screen for a hemoglobin value < 10 go! in pregnancy; and (2) to determine the sensitivity, specificity, positive predictive value and negative predictive value of the copper sulfate test compared with a hemoglobin level obtained by Coulter count.

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2. Methods

According to the nomogram of Van Slyke et al. [9], using the mean plasma protein concentration according to Elrad and Gleicher [lo], an SG was chosen at which to screen antenatal patients for a hemoglobin concentration < 10 g%. At the start of the study, blood was taken for Coulter hemoglobin estimation from all antenatal patients during their first visit to Kalafong Hospital. Initially one of the investigators (L.R.P. or M.F.) used a drop of this blood for hemoglobin assessment by copper sulfate densitometry according to the method of Phillips et al. [8]. Later in the study, a nurse trained by one of the investigators (M.F.) performed the hemoglobin assessment. The copper sulfate method of hemoglobin assessment entailed letting a drop of blood fall into a copper sulfate solution of the correct density and noting whether the drop sank (high hemoglobin), rose (low hemoglobin) or hovered (high hemoglobin) in the copper sulfate solution. The investigators prepared the copper sulfate solution as follows: a laboratory technician at Kalafong Hospital prepared a stock solution (of SG 1.100) from CuS04.5Hz0 crystals according to the protocol of Phillips et al. [8]; one of the investigators (L.R.P. or M.F.) prepared the working solutions by further aqueous dilution of the stock solution. This was tested prospectively on 100 antenatal patients to determine the sensitivity, specificity and positive and negative predictive values of the copper sulfate screening test compared with Coulter hemoglobin estimation. The patients’ gestational ages were also noted to determine whether the test performed equally well in all three trimesters. The hypothesis was that the test should have a sensitivity and specificity of at least 95%, giving a negative predictive value of 99% and positive predictive value of 67% for a prevalence of anemia (Hb < 10 g%) of 10%. 3. Results

A copper sulfate solution with SG 1.045 was initially chosen, according to the nomogram of Van Slyke et al. [9], using the pregnant mean of plasma protein concentration according to Elrad and Gleicher [lo] of 65 g/100 ml (range 55-75 g/100

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ml). The first batch of tests was very discouraging, with all drops of blood sinking. The lowest hemoglobin concentration was 8.4 g%. Several possible reasons for the apparent failure of the test were considered: that a taller container should have been used for the copper sulfate solution, that fresh blood should have been used rather than blood anticoagulated with EDTA, that the mean plasma protein concentration of our patient population differed from that of Elrad and Gleicher [lo], that the nomogram of Van Slyke et al. [9] was not applicable to pregnant patients, and that the SG of our copper sulfate solution was incorrect. The first two factors were changed, but without success. Plasma protein estimations were requested on a sample of 30 consecutive patients in this study, yielding almost exactly the same plasma protein concentration as that of Elrad and Gleicher [lo] (mean 66 g"!~, S.D. 4.3 g%, range 58-75 g%). Pyknometric analysis of the copper sulfate stock solution demonstrated an SG of 1.010 (against an expected SG of 1.100). A stock solution was therefore ordered from Clinical Science Diagnostics (P.O. Box 6157, Halfway House, South Africa 1685, Tel. +27 11 3151146), the company that prepares the copper sulfate solution used by the South African Blood Transfusion Service. This was then diluted to the required concentration according to the method of Phillips et al. [8]. A solution with SG 1.045 (according to the nomogram of Van Slyke et al. [9], adjusted for the lower plasma protein concentration in pregnancy) was initially used, but this seemed to correspond to a hemoglobin concentration lower than 10 g%. A copper sulfate solution with SG 1.048 (according Table 1 Results of 100 consecutive copper sulfate densitometry

patients

Coulter

screened

hemoglobin

for anemia

with

analysis

Hb < 10 g% Hb t 10 g% Total CuSO,-positive CuSO4-negative Total

16 1 17

4 79 83

20 80 100

CuS04-positive, droplet rising; CuS04-negative, droplet sinking or hovering (see text); sensitivity, 94%; specificity, 95%; positive predictive value, 80%; negative predictive value, 99%.

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to the nomogram, without the adjustment for a lower plasma protein concentration) was then tested and seemed to perform better. This solution was therefore then prospectively tested on 100 consecutive patients at their first antenatal visit, yielding the results given in Table 1. Hemoglobin values ranged from 5.5 to 14.9 g%, with a mean of 11.25 g% and a standard deviation of 1.54 g%. There were 17 patients with hemoglobin levels below 10 g%. The copper sulfate test had an accuracy of 95%. There was one false-negative (Hb 9.7 g%) and four false-positive results (Hb 10.0, 11.2, 11.4 and 11.5 g”h). Due to the low error rate, no significant correlation could be detected between erroneous results and gestational age. 4. Discussion

Once the initial technical difficulties had been overcome, this inexpensive test proved simple and accurate to use (although we could not match the 100% sensitivity and specificity of the Jamaican study [7]). The copper sulfate test could therefore be a viable alternative to traditional methods of screening for anemia in pregnancy. Cost-wise, using the copper sulfate solutions makes very good sense. A lOO-ml standard solution should be replaced by a fresh solution after receiving 50 drops of blood, to minimize the error in measurement [8]. This could be ensured by ticking off each drop added to the solution on a piece of paper attached to the container. At a cost of $14 (R 42) for 5 1 of standard copper sulfate solution, the test costs less than 1 US cent per sample. In comparison, hemoglobin estimation by a laboratory costs $1.40 (R 6.20) [ll]. Using a BMS hemoglobinometer requires a capital outlay of $293 (R 880), as well as hemolytic sticks at $4.88 (R 14.64) per 100, in addition to a supply of fresh batteries and a replacement for the glass chamber and clip ($33 or R 99.54) if they get lost or broken. One potential disadvantage of the copper sulfate solution is that the specific hemoglobin level is not known. However this is irrelevant for screening purposes. It is also possible to devise a series of copper sulfate solutions, to obtain a hemoglobin level to the nearest 1 g%, which is the method originally described. It is envisaged that a lower cut-off point could be used to decide which pa-

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tients require further evaluation, such as evaluation of the red cell parameters, ferritin levels, or screening for malaria parasites. Since completion of the study, the method has been implemented with success as routine screening at each patient’s first antenatal visit, and repeated at 28 and 34 weeks of gestation. The nursing staff performing the test have accepted it well due to the immediate availability of the results and the fact that performing the copper sulfate test itself is much quicker than filling the forms required for laboratory estimation of hemoglobin. Each antenatal patient is offered prophylactic iron and folic acid supplements in accordance with WHO guidelines [2]. Patients with a hemoglobin level below 10 g?/ according to the copper sulfate test are offered therapeutic iron and folic acid supplementation, and a full blood count is requested for each of these patients. Prospective evaluation of the protocol, in conjunction with evaluation of the population distribution of hemoglobin in pregnancy in the residents of greater Atteridgeville, is being carried out to determine the level of hemoglobin which should serve as a limit for undertaking further investigation into anemia. This test can be recommended to anyone interested in screening pregnant patients for anemia. Anyone interested is urged to read the articles by Phillips et al. [8] and Van Slyke et al. [9]. A list of requirements and simplified instructions are available from the authors.

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References

111 Letsky EA. Blood volume, haematinics, anaemia. In: De

Swiet M, editor. Medical disorders in obstetric practice. Oxford: Blackwell, 1989: 48-102. t4 World Health Organization. Prevention and management of severe anaemia in pregnancy. Geneva: WHO, 1993. [31 Gjorup T, Bugge PM, Hendricksen C, Jensen AM. A critical evaluation of the clinical diagnosis of anemia. Am J Epidemiol 1986; 124: 657-665. [41 Sanchez-Carillo CL Bias due to conjunctiva hue and the clinical assessment of anaemia. J Clin Epidemiol 1989; 42: 751-754. [51 Linegar AG, Knottenbelt JD, Wormald P-J. Accuracy of a portable haemoglobinometer in clinical practice. S Afr Med J 1991; 79: 547-548. 161 England JM, Walford DM, Waters DAW. Reassessment of the reliability of the haematocrit. Br J Haematol 1972; 23: 247-256.

Stone JE, Simmons WK, Jutsum PJ, Gurney JM. An evaluation of methods of screening for anaemia. Bull WHO 1984; 62: 115-120. 181 Phillips RA, Van Slyke DD, Hamilton PB, Dole VP, Emerson K, Archibald RM. Measurement of specific gravities for whole blood and plasma by standard copper sulfate solutions. J Biol Chem 1950; 183: 305-330. [91 Van Slyke DD, Phillips RA, Dole VP, Hamilton PB, Archibald RM, Plazin J. Calculation of hemoglobin from blood specific gravities. J Biol Chem 1950; 183: [71

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UOI Elrad H, Gleicher N. Physiologic changes in normal pregnancy. In: Gleicher N, editor. Principles of medical therapy in pregnancy. New York: Plenum, 1985: 52. 1111Medical Association of South Africa. Guide to fees for medical services 1995. Pinelands: MASA Publications, 1994: 186.