BRIEF REPORT visual colorimetry, peritoneal lavage fluid
Rapid Visual Colorimetry of Peritoneal Lavage Fluid Study hypothesis: That visual colorimetry can be used to rapidly and precisely estimate the erythrocyte count of 1:5 dilutions of simulated peritoneal lavage fluid. Population: Fifty-four normal adult h u m a n subjects. Methods: The automated or chamber RBC count is often used on fluid obtained by peritoneal lavage in patients with abdominal trauma to help determine the need for surgery. Unfortunately, this method sometimes results in excessive delay. We designed and built a simple colorimeter that facilitated rapid direct visual comparison of u n k n o w n samples w i t h k n o w n color standards. A radiograph view box was used as a light source. Standards were prepared in 16-ram glass tubes to simulate peritoneal lavage fluid with RBC counts ranging from 0 to 140,000 in 10,000 cell/~L increments; 1:5 dilutions with water were used throughout to reduce opacity. Thimerosal was added to unknowns and standards to stabilize color; all samples were kept refrigerated at 4 C when not in use. In a double-blind in-vitro study, each subject matched 20 randomly distributed unknowns ranging from 12,000 to 131,000 erythrocytes/b~L to the nearest standard. Results: The mean absolute error for all 1,080 determinations was 3,560 RBC/b~L (95% CI=4,290-4,830; SD=4,560; t=39.6; dr=l,079; P < .001). This method correctly predicted the RBC count to within 9,000 cells/bLL 95% of the time. Conclusion: Visual comparison of 1:5 dilutions of simulated peritoneal lavage fluid with known color standards can be used to rapidly and precisely estimate the erythrocyte count. [Tandberg D, Reitmeyer ST, Cheney PR: Rapid visual colorimetry of peritoneal lavage fluid. Ann Emerg Med November 1990;19:1318-132i.]
Dan Tandberg, MD, FACEP Sheila T Reitmeyer, BS Paul R Cheney, MD Albuquerque, New Mexico From the Division of Emergency Medicine, University of New Mexico School of Medicine, Albuquerque. Received for publication November 20, 1989. Revision received April 2, 1990. Accepted for publication June 14, 1990. This work was supported in part by the Dean's Medical Student Research Fund, University of New Mexico School of Medicine. Address for reprints: Dan Tandberg, MD, Division of Emergency Medicine, University of New Mexico School of Medicine, 620 Camino de Salud NE, Albuquerque, New Mexico 87131.
INTRODUCTION Trauma is a major source of death in young people, and abdominal injuries occur in many of these patients. Unfortunately, physical diagnosis is often misleading in those with intra-abdominal injury. Emergency physicians and trauma surgeons at present rely on clinical laboratory determinations, especially the RBC count, on fluid obtained by peritoneal lavage to help select those who need surgery, but in some cases this takes longer than is desirable. Visual colorimetry is a very old method for estimating the concentration of colored substances in solution. We conducted the following double-blind, in-vitro study in order to measure the accuracy of visual colorimetry and assess its potential usefulness as a rapid bedside method of estimating the RBC count in peritoneal lavage fluid. METHODS A simple visual colorimeter 1 was designed and produced in a home woodworking shop (Figure 1). This device permits direct comparison of pairs of glass tubes containing colored fluids, while excluding distracting room light and reducing reflections. A stock solution of simulated peritoneal lavage fluid was prepared as follows: Whole blood was obtained in 7-mL Vacutainer ® tubes containing EDTA (Becton Dickinson, Rutherford, New Jersey) from a single healthy
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FIGURE 1. Photograph of the visual colorimeter in use. The person evaluating the u n k n o w n sample holds the device about 12 in. from the radiograph view box and is unaware of which standard she is being shown. v o l u n t e e r donor k n o w n to be HIV-1 negative, at l o w risk for AIDS, and w i l l i n g to d o n a t e p e r i o d i c a l l y . T h e CBC was d e t e r m i n e d in t r i p l i c a t e u s i n g a C o u l t e r ® M o d e l S-Plus IV multiparameter automated impedance type cell counter (Coulter Electronics, Hialeah, Florida). The m e a n RBC count was 5.67 x 106/~xL; hemoglobin, 17.0 g/dL; and m e a n corpuscular h e m o g l o b i n (MCH), 30.0 pg. The n o r m a l adult m a l e m e a n RBC count is 5.4 x 106/~L. To s i m u l a t e peritoneal lavage fluid w i t h an RBC count of IO0,O00/~L, a 1:54 dilution is required: l O0,O00/ixL 1,000 mL × - 18.52 mL 5.40 × 106/~L T h i s v o l u m e t h e n was n o r m a l i z e d u s i n g t h e r a t i o of t h e m e a n a d u l t m a l e and donor RBC values: 5.40 x 106/txL x 18.52 mL 5.67 x 106/ixL
17.64 mL
Thus, the s t o c k solution was actually prepared by diluting 17.64 mL of this donor's blood to 1L w i t h n o r m a l saline. This fluid, however, is opaque and does not t r a n s m i t enough light to be visually evaluated accurately. A deriv a t i o n of the Beer-Lambert law pred i c t s t h a t t h e m i n i m u m e r r o r in m e a s u r e m e n t of c o n c e n t r a t i o n of a colored solute occurs at a light transm i t t a n c e of 0.368. 2 P r e l i m i n a r y exp e r i m e n t s s h o w e d that a fivefold dil u t i o n of peritoneal lavage fluid w i t h a c e l l c o u n t of 100,000 w o u l d be close to this o p t i m u m . We therefore chose to dilute all standard and unk n o w n samples 1:5. A set of 15 color comparison standards was prepared corresponding to 1:5 dilutions of peritoneal fluid w i t h cell counts ranging from 0 to 140,000/txL. T h e d i l u e n t s o l u t i o n consisted of 100 m g of t h i m e r o s a l dil u t e d to a v o l u m e of 1 L w i t h distilled water. Each standard was prepared by m i x i n g the appropriate volu m e of s t o c k b l o o d s o l u t i o n w i t h e n o u g h d i l u e n t to p r o d u c e a t o t a l v o l u m e of 10 mL. S t a n d a r d s w e r e placed in 16 m m by 100 m m borosilic a t e glass d i s p o s a b l e c u l t u r e t u b e s 134/1319
(Helps P r o d u c t s Inc, F r a n k l i n , Wisconsin), s e a l e d w i t h y e l l o w p l a s t i c caps ("Ezee T o p p e r s , " Baxter Scientific Products, Irvine, California), and l a b e l e d w i t h t h e i r p r e d i l u t i o n RBC count. A set of 20 u n k n o w n samples was prepared in a s i m i l a r fashion. Thirt e e n of t h e s e r a n g e d f r o m RBC counts b e t w e e n 10,000 and 140,000; an additional seven u n k n o w n s varied b e t w e e n 9 0 , 0 0 0 a n d 110,000. To avoid bias, the values of u n k n o w n s were m a d e to deviate from standards rather t h a n to m a t c h t h e m exactly; a table of r a n d o m n u m b e r s was used to generate t h e actual values. W h e n not in use, standards and u n k n o w n s were refrigerated at 4 C. A c o n v e n i e n c e s a m p l e of h e a l t h y adult h u m a n subjects was presented w i t h a r a n d o m s e q u e n c e of the 20 u n k n o w n samples and were asked to match them with color standards. Subjects were excluded if t h e y were over 55 years of age, unable to read n e w s p r i n t a t a d i s t a n c e of n i n e i n c h e s w i t h o u t c o r r e c t i v e lenses, or color blind. A radiograph view box provided a consistent, readily available light source; the c o l o r i m e t e r was held app r o x i m a t e l y 30 c m (12 inches) from t h e v i e w b o x for all c o m p a r i s o n s . T h e c o d e s of t h e u n k n o w n t u b e s were covered so that n e i t h e r the subject nor the recorder k n e w the count. Subjects were asked to m a t c h each Annals of Emergency Medicine
u n k n o w n w i t h its m o s t s i m i l a r appearing standard. Tubes were m i x e d by inversion before each test. The exp e r i m e n t e r placed an u n k n o w n tube into one cell of the c o l o r i m e t e r and began by inserting an obviously m o r e dilute standard tube for comparison. The subject was then asked to report if t h e s t a n d a r d w a s " l i g h t e r , " " d a r k e r , " or " t h e s a m e " as the unknown. This was carried out with standards of increasing c o n c e n t r a t i o n u n t i l t h e t u b e s a p p e a r e d to m a t c h , and this value was noted. This process was repeated, beginn i n g w i t h a s t a n d a r d several t u b e s darker t h a n the u n k n o w n , comparing w i t h s t a n d a r d s of d i m i n i s h i n g conc e n t r a t i o n . If t h e s u b j e c t chose the same standard on the ascending and descending trials, this value was recorded. If the subject had d i f f i c u l t y choosing between two adjacent tubes, t h e s e were p r e s e n t e d repeate d l y u n t i l an u n a m b i g u o u s c h o i c e w a s m a d e . If t w o s t a n d a r d t u b e s s e e m e d a n e q u a l l y good m a t c h , the m i d p o i n t of t h e i r v a l u e s w a s recorded. T h e error of each e s t i m a t i o n was d e t e r m i n e d by s u b t r a c t i n g each unknown's value from the colori m e t r i c a l l y e s t i m a t e d one and obtaining the absolute value of this difference. The hypothesis that the m e a n a b s o l u t e error of c o l o r i m e t r i c estimations was zero was tested using Student's t d i s t r i b u t i o n (paired, 19:11 November1990
FIGURE 2. Frequency distribution of the measurement errors associated with 1,080 in-vitro RBC estimations in simulated peritoneal lavage fluid. Error is in thousands of cells per ~L.
150
I
mations were typically carried out in one to two minutes, but this was not formally evaluated.
N = 1,080
100
O ¢@ CY 1.1..
50
-20
-15
-10
-5
0
5
10
15
20
Error (in thousands) 2 two-tailed). Linear r e g r e s s i o n was used to test for association of absolute error with u n k n o w n cell counts. An c~ of .05 was used throughout. N e w sets of standards were prepared at m o n t h l y intervals and stored in a refrigerator at 4 C over a fourm o n t h period to assess their stability. A single set of standards was u s e d c o n s i s t e n t l y t h r o u g h o u t the study. Prior approval by our institution's H u m a n Research Review Committee was obtained; informed consent was given by each subject. RESULTS T w e n t y - s i x m a l e and 28 f e m a l e
19:11November1990
subjects (mean age, 30 years; range, 15 to 52) were entered and completed the study. T h e m e a n absolute error for all 1,080 tests was 3,560 RBC/~L, significantly differently f r o m zero (8D, 4,560; 95% CI, 4,290-4,830; t, 39.6; df, 1,079; P < .001). In only 45 m e a s u r e m e n t s (4.2%) was the error more than 9,000 cells, and in only one instance (0.09%) was an error of more than 15,000 cells recorded (Figure 2). The mean absolute error did not vary significantly with increasing u n k n o w n cell c o u n t (R 2, 0.22; P > .05). Standards prepared at m o n t h l y i n tervals remained stable in color over a four-month period. Individual estiAnnals of Emergency Medicine
DISCUSSION Before 1965, a b d o m i n a l paracentesis was used in patients with blunt abdominal t r a u m a to help ascertain the need for surgery. The withdrawal of 20 mL or more of frank blood was considered a positive test and mandated laparotomy. However, this was insensitive; only one fifth of patients needing emergency surgical intervention had a positive result. In 1965, Root described the techn i q u e of d i a g n o s t i c p e r i t o n e a l lavage. 3 Visual inspection of the recovered fluid was used to evaluate the patient's likelihood of serious intraabdominal injury. By using the criterion of "the presence of more than a faint salmon-pink tinge of blood in the retrieved p e r f u s a t e , " these authors accurately differentiated those who needed operation from those who did not in 28 consecutive cases. As the use of this technique became widespread, however, it became apparent that m a n y patients underwent unnecessary laparotomy if Root's criteria for a positive test were used. Caffee tried to develop a more quantitative a p p r o a c h by p u t t i n g small volumes of blood into 1-L bottles of n o r m a l saline and using these for comparison. 4 0 l s o n described the refinement of attempting to read newsprint through the IV tubing, s but this also turned out to be inaccurate. As automated RBC counts became available, physicians gradually adopted them. Blunt abdominal trauma patients with 100,000 erythrocytes/~L or more were considered to have a positive test and to require surgery. Estimates of the diagnostic accuracy of peritoneal lavage evaluated with laboratory erythrocyte c o u n t s h a v e r a n g e d f r o m 90% to 99%. Today's physicians rely almost universally on a u t o m a t e d or c h a m b e r l a b o r a t o r y RBC c o u n t s , a l t h o u g h there is some disagreement about the cell count best used as the threshold for a positive test. A problem w i t h laboratory counts is that they some-
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t i m e s t a k e too long (29 m i n u t e s in our laboratory, w i t h a range of 17 to 51 minutes). We were convinced that a m o r e r a p i d m e t h o d of e s t i m a t i o n w o u l d be w e l c o m e d by m a n y clinicians. V i s u a l c o l o r i m e t r y o r i g i n a t e d in the sugar, dye, and w i n e m a k i n g ind u s t r i e s . It w a s f i r s t d e s c r i b e d in 18276 a n d w a s u s e d in e a r l y biochemical assays, including hemoglobin. 7 However, t y p i c a l u n d i l u t e d peritoneal lavage fluid in the range of c l i n i c a l i n t e r e s t does n o t t r a n s m i t enough light to 'be reliably evaluated w i t h this m e t h o d . We used a fivefold dilution of standards and u n k n o w n s to achieve a light t r a n s m i t t a n c e close to the o p t i m u m of 0.368. 2 The overall dilution used in this study, 1:270 (1:54 followed by 1:5), is quite similar to the 1:251 dilution used in older laboratory assays of h e m o g l o b i n concentration. We chose distilled w a t e r as the dil u e n t in order to h e m o l y z e the red cells, release free hemoglobin, and reduce t u r b i d i t y . We n o r m a l i z e d t h e d o n o r ' s a c t u a l RBC c o u n t to t h e m e a n for m e n in order to increase acc u r a c y of t h e color standards. (It is important that the individual who donates blood for the s t a n d a r d s be h e m a t o l o g i c a l l y normal. Ideally, the M C H should be 30.0 pg; small deviations from this value w o u l d lead to m o d e s t s y s t e m a t i c error.) T h e d e v e l o p m e n t of s t a b l e c o l o r c o m p a r i s o n s t a n d a r d s was difficult. We a t t e m p t e d to m a t c h the color of hemoglobin with various commerc i a l l y a v a i l a b l e i n k s a n d dyes b u t were unsuccessful. Earlier techniques using hemoglobin stabilized with carbon monoxide, cyanide, or hydrochloric acid were n o t d e e m e d rapid or safe enough for bedside use in an acute trauma care setting. Fortunately, the a d d i t i o n of t h i m e r o s a l yielded color standards that maint a i n e d a c o n s t a n t h u e and r e s i s t e d b a c t e r i a l d e g r a d a t i o n o v e r a four-
136/1321
m o n t h period, if k e p t refrigerated. We f o u n d t h a t subjects were able to m a t c h u n k n o w n s to t h e closest s t a n d a r d t u b e u s i n g the p r o t o t y p e c o l o r i m e t e r w i t h a surprising degree of precision and accuracy. The m e a n absolute error found (3,560 RBC/txL) was significantly different from zero; this r e p r e s e n t s c u m u l a t i v e i m p r e c i sion in p i p e t t i n g and in the original a u t o m a t e d e s t i m a t i o n of t h e donor RBC count. The small m a g n i t u d e of this error c o m p a r e d w i t h the values being m e a s u r e d m a k e it u n l i k e l y to be c l i n i c a l l y misleading. The coefficient of variation for our method (5.5%)compares favorably with published estimates for e r y t h r o c y t e counts using the counting c h a m b e r m e t h o d (CV 7.7%).8 The reported precision of a u t o m a t e d techniques used by m o s t clinical laboratories (CV = 0.9% to 3%) are good for samples w i t h RBC counts in the normal range. 9 However, studies q u a n t i f y i n g t h e p r e c i s i o n of a u t o m a t e d e r y t h r o c y t e c o u n t s in t h e range of 100,000 cells are lacking. Ext r a p o l a t i o n of data f r o m a u t o m a t e d platelet counts in t h r o m b o c y t o p e n i c patients suggests that the accuracy of a u t o m a t e d RBC counts in the range of 100,000 cells/vtL m a y be similar to that found w i t h our m e t h o d . ~o C o l o r i m e t r i c e s t i m a t i o n s by o u r s u b j e c t s u s u a l l y r e q u i r e d less t h a n two m i n u t e s . Preparation of the 1:5 d i l u t i o n of lavage fluid from actual patients w o u l d be expected to add no more than one to two m i n u t e s to the total t i m e for the estimation, but this w a s n o t m e a s u r e d in t h e c u r r e n t study. F u r t h e r s t u d y w i l l be required to d e t e r m i n e if t h i s m e t h o d c o u l d be m o d i f i e d for use in c e n t e r s w h e r e RBC c o u n t s of 5 0 , 0 0 0 c e l l s a n d higher are considered positive. CONCLUSION Visual c o l o r i m e t r y can be used to rapidly and a c c u r a t e l y e s t i m a t e the
Annals of Emergency Medicine
RBC c o u n t of s i m u l a t e d p e r i t o n e a l lavage fluid. A c o m p a r a t i v e i n - v i v o study will be needed to evaluate the u s e f u l n e s s of t h i s m e t h o d in t r a u m a t i z e d patients. Details for construction of the colorimeter prototype and for preparation of color comparison standards are available on request from Dr Tandberg.
The authors gratefully acknowledge assistance with manuscript preparation by Joanne Snowdon and Sandra Mirabal. The photograph and illustration were prepared by Delilah Montoya, Brian Degani, and Denis McKeon. Beulah M Woodfin and Jimmy C Standefer graciously provided technical assistance and guidance.
REFERENCES
1. Hawk PB, Olaf B: Practical Physiological Chemistry. ed 9. Philadelphia, Blakiston's Son
& Co, 1926, p 24, 361. 2. Pecsok RL, Shields LD: Quantitative analysis by absorption of electromagnetic radiation, in Moderr~ Methods of Chemical Analysis. New York, John Wiley and Sons, Ine, 1968, p 147-161. 3. Root HD, Hauser CW, McKinley CR, et al: Diagnostic peritoneal lavage. Surgery 1965;57: 633-637. 4. Caffee HH, Benfield JR: ts peritoneal lavage for the diagnosis of hemoperitoneum safe? Arch Surg 1971;103:4-7. 5. Olsen WR, Redman HC, Hildreth DH: Quantitative peritoneal lavage in blunt abdominal trauma. Arch Surg 1972;104:536-543. 6. HoutoukLabillardiere JJ: Description d'un colorimetre: Extrait des acres de l'Academie RoyaIe des Sciences. Belles-Lettres et Arts de Rouen. Rouen, France, 1827.
7. Todd JC, Sanford AH: Hemoglobin, in Clirlical Diagnosis by Laboratory Methods, ed 6.
Philadelphia, WB Saunders Co, 1927, p 232-241. 8. Nelson DA: Basic Methodology, in Henry JB (ed): Clinical Diagnosis and Management by Laboratory Methods, ed 16. Philadelphia, WB Saunders Co, 1979, p 858-917. 9. Koepke JA, Protextor TJ: Quality assurance for multichannel hematology instruments. Am J Clin PathoI 1981;75:28-33. 10. Brittin GM, Dew SA, Fewell EK: Automated optical counting of blood platelets. Blood 1971;38:422-430.
19:11 November 1990
Rapid Visual Colorimetry of Peritoneal Lavage Fluid Study hypothesis: That visual colorimetry can be used to rapidly and precisely estimate the erythrocyte count of 1:5 dilutions of simulated peritoneal lavage fluid. Population: Fifty-four normal adult h u m a n subjects. Methods: The automated or chamber RBC count is often used on fluid obtained by peritoneal lavage in patients with abdominal trauma to help determine the need for surgery. Unfortunately, this method sometimes results in excessive delay. We designed and built a simple colorimeter that facilitated rapid direct visual comparison of u n k n o w n samples w i t h k n o w n color standards. A radiograph view box was used as a light source. Standards were prepared in 16-ram glass tubes to simulate peritoneal lavage fluid with RBC counts ranging from 0 to 140,000 in 10,000 cell/~L increments; 1:5 dilutions with water were used throughout to reduce opacity. Thimerosal was added to unknowns and standards to stabilize color; all samples were kept refrigerated at 4 C when not in use. In a double-blind in-vitro study, each subject matched 20 randomly distributed unknowns ranging from 12,000 to 131,000 erythrocytes/b~L to the nearest standard. Results: The mean absolute error for all 1,080 determinations was 3,560 RBC/b~L (95% CI=4,290-4,830; SD=4,560; t=39.6; dr=l,079; P < .001). This method correctly predicted the RBC count to within 9,000 cells/bLL 95% of the time. Conclusion: Visual comparison of 1:5 dilutions of simulated peritoneal lavage fluid with known color standards can be used to rapidly and precisely estimate the erythrocyte count. [Tandberg D, Reitmeyer ST, Cheney PR: Rapid visual colorimetry of peritoneal lavage fluid. Ann Emerg Med November 1990;19:1318-132i.]
Dan Tandberg, MD, FACEP Sheila T Reitmeyer, BS Paul R Cheney, MD Albuquerque, New Mexico From the Division of Emergency Medicine, University of New Mexico School of Medicine, Albuquerque. Received for publication November 20, 1989. Revision received April 2, 1990. Accepted for publication June 14, 1990. This work was supported in part by the Dean's Medical Student Research Fund, University of New Mexico School of Medicine. Address for reprints: Dan Tandberg, MD, Division of Emergency Medicine, University of New Mexico School of Medicine, 620 Camino de Salud NE, Albuquerque, New Mexico 87131.
INTRODUCTION Trauma is a major source of death in young people, and abdominal injuries occur in many of these patients. Unfortunately, physical diagnosis is often misleading in those with intra-abdominal injury. Emergency physicians and trauma surgeons at present rely on clinical laboratory determinations, especially the RBC count, on fluid obtained by peritoneal lavage to help select those who need surgery, but in some cases this takes longer than is desirable. Visual colorimetry is a very old method for estimating the concentration of colored substances in solution. We conducted the following double-blind, in-vitro study in order to measure the accuracy of visual colorimetry and assess its potential usefulness as a rapid bedside method of estimating the RBC count in peritoneal lavage fluid. METHODS A simple visual colorimeter 1 was designed and produced in a home woodworking shop (Figure 1). This device permits direct comparison of pairs of glass tubes containing colored fluids, while excluding distracting room light and reducing reflections. A stock solution of simulated peritoneal lavage fluid was prepared as follows: Whole blood was obtained in 7-mL Vacutainer ® tubes containing EDTA (Becton Dickinson, Rutherford, New Jersey) from a single healthy
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FIGURE 1. Photograph of the visual colorimeter in use. The person evaluating the u n k n o w n sample holds the device about 12 in. from the radiograph view box and is unaware of which standard she is being shown. v o l u n t e e r donor k n o w n to be HIV-1 negative, at l o w risk for AIDS, and w i l l i n g to d o n a t e p e r i o d i c a l l y . T h e CBC was d e t e r m i n e d in t r i p l i c a t e u s i n g a C o u l t e r ® M o d e l S-Plus IV multiparameter automated impedance type cell counter (Coulter Electronics, Hialeah, Florida). The m e a n RBC count was 5.67 x 106/~xL; hemoglobin, 17.0 g/dL; and m e a n corpuscular h e m o g l o b i n (MCH), 30.0 pg. The n o r m a l adult m a l e m e a n RBC count is 5.4 x 106/~L. To s i m u l a t e peritoneal lavage fluid w i t h an RBC count of IO0,O00/~L, a 1:54 dilution is required: l O0,O00/ixL 1,000 mL × - 18.52 mL 5.40 × 106/~L T h i s v o l u m e t h e n was n o r m a l i z e d u s i n g t h e r a t i o of t h e m e a n a d u l t m a l e and donor RBC values: 5.40 x 106/txL x 18.52 mL 5.67 x 106/ixL
17.64 mL
Thus, the s t o c k solution was actually prepared by diluting 17.64 mL of this donor's blood to 1L w i t h n o r m a l saline. This fluid, however, is opaque and does not t r a n s m i t enough light to be visually evaluated accurately. A deriv a t i o n of the Beer-Lambert law pred i c t s t h a t t h e m i n i m u m e r r o r in m e a s u r e m e n t of c o n c e n t r a t i o n of a colored solute occurs at a light transm i t t a n c e of 0.368. 2 P r e l i m i n a r y exp e r i m e n t s s h o w e d that a fivefold dil u t i o n of peritoneal lavage fluid w i t h a c e l l c o u n t of 100,000 w o u l d be close to this o p t i m u m . We therefore chose to dilute all standard and unk n o w n samples 1:5. A set of 15 color comparison standards was prepared corresponding to 1:5 dilutions of peritoneal fluid w i t h cell counts ranging from 0 to 140,000/txL. T h e d i l u e n t s o l u t i o n consisted of 100 m g of t h i m e r o s a l dil u t e d to a v o l u m e of 1 L w i t h distilled water. Each standard was prepared by m i x i n g the appropriate volu m e of s t o c k b l o o d s o l u t i o n w i t h e n o u g h d i l u e n t to p r o d u c e a t o t a l v o l u m e of 10 mL. S t a n d a r d s w e r e placed in 16 m m by 100 m m borosilic a t e glass d i s p o s a b l e c u l t u r e t u b e s 134/1319
(Helps P r o d u c t s Inc, F r a n k l i n , Wisconsin), s e a l e d w i t h y e l l o w p l a s t i c caps ("Ezee T o p p e r s , " Baxter Scientific Products, Irvine, California), and l a b e l e d w i t h t h e i r p r e d i l u t i o n RBC count. A set of 20 u n k n o w n samples was prepared in a s i m i l a r fashion. Thirt e e n of t h e s e r a n g e d f r o m RBC counts b e t w e e n 10,000 and 140,000; an additional seven u n k n o w n s varied b e t w e e n 9 0 , 0 0 0 a n d 110,000. To avoid bias, the values of u n k n o w n s were m a d e to deviate from standards rather t h a n to m a t c h t h e m exactly; a table of r a n d o m n u m b e r s was used to generate t h e actual values. W h e n not in use, standards and u n k n o w n s were refrigerated at 4 C. A c o n v e n i e n c e s a m p l e of h e a l t h y adult h u m a n subjects was presented w i t h a r a n d o m s e q u e n c e of the 20 u n k n o w n samples and were asked to match them with color standards. Subjects were excluded if t h e y were over 55 years of age, unable to read n e w s p r i n t a t a d i s t a n c e of n i n e i n c h e s w i t h o u t c o r r e c t i v e lenses, or color blind. A radiograph view box provided a consistent, readily available light source; the c o l o r i m e t e r was held app r o x i m a t e l y 30 c m (12 inches) from t h e v i e w b o x for all c o m p a r i s o n s . T h e c o d e s of t h e u n k n o w n t u b e s were covered so that n e i t h e r the subject nor the recorder k n e w the count. Subjects were asked to m a t c h each Annals of Emergency Medicine
u n k n o w n w i t h its m o s t s i m i l a r appearing standard. Tubes were m i x e d by inversion before each test. The exp e r i m e n t e r placed an u n k n o w n tube into one cell of the c o l o r i m e t e r and began by inserting an obviously m o r e dilute standard tube for comparison. The subject was then asked to report if t h e s t a n d a r d w a s " l i g h t e r , " " d a r k e r , " or " t h e s a m e " as the unknown. This was carried out with standards of increasing c o n c e n t r a t i o n u n t i l t h e t u b e s a p p e a r e d to m a t c h , and this value was noted. This process was repeated, beginn i n g w i t h a s t a n d a r d several t u b e s darker t h a n the u n k n o w n , comparing w i t h s t a n d a r d s of d i m i n i s h i n g conc e n t r a t i o n . If t h e s u b j e c t chose the same standard on the ascending and descending trials, this value was recorded. If the subject had d i f f i c u l t y choosing between two adjacent tubes, t h e s e were p r e s e n t e d repeate d l y u n t i l an u n a m b i g u o u s c h o i c e w a s m a d e . If t w o s t a n d a r d t u b e s s e e m e d a n e q u a l l y good m a t c h , the m i d p o i n t of t h e i r v a l u e s w a s recorded. T h e error of each e s t i m a t i o n was d e t e r m i n e d by s u b t r a c t i n g each unknown's value from the colori m e t r i c a l l y e s t i m a t e d one and obtaining the absolute value of this difference. The hypothesis that the m e a n a b s o l u t e error of c o l o r i m e t r i c estimations was zero was tested using Student's t d i s t r i b u t i o n (paired, 19:11 November1990
FIGURE 2. Frequency distribution of the measurement errors associated with 1,080 in-vitro RBC estimations in simulated peritoneal lavage fluid. Error is in thousands of cells per ~L.
150
I
mations were typically carried out in one to two minutes, but this was not formally evaluated.
N = 1,080
100
O ¢@ CY 1.1..
50
-20
-15
-10
-5
0
5
10
15
20
Error (in thousands) 2 two-tailed). Linear r e g r e s s i o n was used to test for association of absolute error with u n k n o w n cell counts. An c~ of .05 was used throughout. N e w sets of standards were prepared at m o n t h l y intervals and stored in a refrigerator at 4 C over a fourm o n t h period to assess their stability. A single set of standards was u s e d c o n s i s t e n t l y t h r o u g h o u t the study. Prior approval by our institution's H u m a n Research Review Committee was obtained; informed consent was given by each subject. RESULTS T w e n t y - s i x m a l e and 28 f e m a l e
19:11November1990
subjects (mean age, 30 years; range, 15 to 52) were entered and completed the study. T h e m e a n absolute error for all 1,080 tests was 3,560 RBC/~L, significantly differently f r o m zero (8D, 4,560; 95% CI, 4,290-4,830; t, 39.6; df, 1,079; P < .001). In only 45 m e a s u r e m e n t s (4.2%) was the error more than 9,000 cells, and in only one instance (0.09%) was an error of more than 15,000 cells recorded (Figure 2). The mean absolute error did not vary significantly with increasing u n k n o w n cell c o u n t (R 2, 0.22; P > .05). Standards prepared at m o n t h l y i n tervals remained stable in color over a four-month period. Individual estiAnnals of Emergency Medicine
DISCUSSION Before 1965, a b d o m i n a l paracentesis was used in patients with blunt abdominal t r a u m a to help ascertain the need for surgery. The withdrawal of 20 mL or more of frank blood was considered a positive test and mandated laparotomy. However, this was insensitive; only one fifth of patients needing emergency surgical intervention had a positive result. In 1965, Root described the techn i q u e of d i a g n o s t i c p e r i t o n e a l lavage. 3 Visual inspection of the recovered fluid was used to evaluate the patient's likelihood of serious intraabdominal injury. By using the criterion of "the presence of more than a faint salmon-pink tinge of blood in the retrieved p e r f u s a t e , " these authors accurately differentiated those who needed operation from those who did not in 28 consecutive cases. As the use of this technique became widespread, however, it became apparent that m a n y patients underwent unnecessary laparotomy if Root's criteria for a positive test were used. Caffee tried to develop a more quantitative a p p r o a c h by p u t t i n g small volumes of blood into 1-L bottles of n o r m a l saline and using these for comparison. 4 0 l s o n described the refinement of attempting to read newsprint through the IV tubing, s but this also turned out to be inaccurate. As automated RBC counts became available, physicians gradually adopted them. Blunt abdominal trauma patients with 100,000 erythrocytes/~L or more were considered to have a positive test and to require surgery. Estimates of the diagnostic accuracy of peritoneal lavage evaluated with laboratory erythrocyte c o u n t s h a v e r a n g e d f r o m 90% to 99%. Today's physicians rely almost universally on a u t o m a t e d or c h a m b e r l a b o r a t o r y RBC c o u n t s , a l t h o u g h there is some disagreement about the cell count best used as the threshold for a positive test. A problem w i t h laboratory counts is that they some-
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VISUAL COLORIMETRY Tandberg, Reitmeyer & Cheney
t i m e s t a k e too long (29 m i n u t e s in our laboratory, w i t h a range of 17 to 51 minutes). We were convinced that a m o r e r a p i d m e t h o d of e s t i m a t i o n w o u l d be w e l c o m e d by m a n y clinicians. V i s u a l c o l o r i m e t r y o r i g i n a t e d in the sugar, dye, and w i n e m a k i n g ind u s t r i e s . It w a s f i r s t d e s c r i b e d in 18276 a n d w a s u s e d in e a r l y biochemical assays, including hemoglobin. 7 However, t y p i c a l u n d i l u t e d peritoneal lavage fluid in the range of c l i n i c a l i n t e r e s t does n o t t r a n s m i t enough light to 'be reliably evaluated w i t h this m e t h o d . We used a fivefold dilution of standards and u n k n o w n s to achieve a light t r a n s m i t t a n c e close to the o p t i m u m of 0.368. 2 The overall dilution used in this study, 1:270 (1:54 followed by 1:5), is quite similar to the 1:251 dilution used in older laboratory assays of h e m o g l o b i n concentration. We chose distilled w a t e r as the dil u e n t in order to h e m o l y z e the red cells, release free hemoglobin, and reduce t u r b i d i t y . We n o r m a l i z e d t h e d o n o r ' s a c t u a l RBC c o u n t to t h e m e a n for m e n in order to increase acc u r a c y of t h e color standards. (It is important that the individual who donates blood for the s t a n d a r d s be h e m a t o l o g i c a l l y normal. Ideally, the M C H should be 30.0 pg; small deviations from this value w o u l d lead to m o d e s t s y s t e m a t i c error.) T h e d e v e l o p m e n t of s t a b l e c o l o r c o m p a r i s o n s t a n d a r d s was difficult. We a t t e m p t e d to m a t c h the color of hemoglobin with various commerc i a l l y a v a i l a b l e i n k s a n d dyes b u t were unsuccessful. Earlier techniques using hemoglobin stabilized with carbon monoxide, cyanide, or hydrochloric acid were n o t d e e m e d rapid or safe enough for bedside use in an acute trauma care setting. Fortunately, the a d d i t i o n of t h i m e r o s a l yielded color standards that maint a i n e d a c o n s t a n t h u e and r e s i s t e d b a c t e r i a l d e g r a d a t i o n o v e r a four-
136/1321
m o n t h period, if k e p t refrigerated. We f o u n d t h a t subjects were able to m a t c h u n k n o w n s to t h e closest s t a n d a r d t u b e u s i n g the p r o t o t y p e c o l o r i m e t e r w i t h a surprising degree of precision and accuracy. The m e a n absolute error found (3,560 RBC/txL) was significantly different from zero; this r e p r e s e n t s c u m u l a t i v e i m p r e c i sion in p i p e t t i n g and in the original a u t o m a t e d e s t i m a t i o n of t h e donor RBC count. The small m a g n i t u d e of this error c o m p a r e d w i t h the values being m e a s u r e d m a k e it u n l i k e l y to be c l i n i c a l l y misleading. The coefficient of variation for our method (5.5%)compares favorably with published estimates for e r y t h r o c y t e counts using the counting c h a m b e r m e t h o d (CV 7.7%).8 The reported precision of a u t o m a t e d techniques used by m o s t clinical laboratories (CV = 0.9% to 3%) are good for samples w i t h RBC counts in the normal range. 9 However, studies q u a n t i f y i n g t h e p r e c i s i o n of a u t o m a t e d e r y t h r o c y t e c o u n t s in t h e range of 100,000 cells are lacking. Ext r a p o l a t i o n of data f r o m a u t o m a t e d platelet counts in t h r o m b o c y t o p e n i c patients suggests that the accuracy of a u t o m a t e d RBC counts in the range of 100,000 cells/vtL m a y be similar to that found w i t h our m e t h o d . ~o C o l o r i m e t r i c e s t i m a t i o n s by o u r s u b j e c t s u s u a l l y r e q u i r e d less t h a n two m i n u t e s . Preparation of the 1:5 d i l u t i o n of lavage fluid from actual patients w o u l d be expected to add no more than one to two m i n u t e s to the total t i m e for the estimation, but this w a s n o t m e a s u r e d in t h e c u r r e n t study. F u r t h e r s t u d y w i l l be required to d e t e r m i n e if t h i s m e t h o d c o u l d be m o d i f i e d for use in c e n t e r s w h e r e RBC c o u n t s of 5 0 , 0 0 0 c e l l s a n d higher are considered positive. CONCLUSION Visual c o l o r i m e t r y can be used to rapidly and a c c u r a t e l y e s t i m a t e the
Annals of Emergency Medicine
RBC c o u n t of s i m u l a t e d p e r i t o n e a l lavage fluid. A c o m p a r a t i v e i n - v i v o study will be needed to evaluate the u s e f u l n e s s of t h i s m e t h o d in t r a u m a t i z e d patients. Details for construction of the colorimeter prototype and for preparation of color comparison standards are available on request from Dr Tandberg.
The authors gratefully acknowledge assistance with manuscript preparation by Joanne Snowdon and Sandra Mirabal. The photograph and illustration were prepared by Delilah Montoya, Brian Degani, and Denis McKeon. Beulah M Woodfin and Jimmy C Standefer graciously provided technical assistance and guidance.
REFERENCES
1. Hawk PB, Olaf B: Practical Physiological Chemistry. ed 9. Philadelphia, Blakiston's Son
& Co, 1926, p 24, 361. 2. Pecsok RL, Shields LD: Quantitative analysis by absorption of electromagnetic radiation, in Moderr~ Methods of Chemical Analysis. New York, John Wiley and Sons, Ine, 1968, p 147-161. 3. Root HD, Hauser CW, McKinley CR, et al: Diagnostic peritoneal lavage. Surgery 1965;57: 633-637. 4. Caffee HH, Benfield JR: ts peritoneal lavage for the diagnosis of hemoperitoneum safe? Arch Surg 1971;103:4-7. 5. Olsen WR, Redman HC, Hildreth DH: Quantitative peritoneal lavage in blunt abdominal trauma. Arch Surg 1972;104:536-543. 6. HoutoukLabillardiere JJ: Description d'un colorimetre: Extrait des acres de l'Academie RoyaIe des Sciences. Belles-Lettres et Arts de Rouen. Rouen, France, 1827.
7. Todd JC, Sanford AH: Hemoglobin, in Clirlical Diagnosis by Laboratory Methods, ed 6.
Philadelphia, WB Saunders Co, 1927, p 232-241. 8. Nelson DA: Basic Methodology, in Henry JB (ed): Clinical Diagnosis and Management by Laboratory Methods, ed 16. Philadelphia, WB Saunders Co, 1979, p 858-917. 9. Koepke JA, Protextor TJ: Quality assurance for multichannel hematology instruments. Am J Clin PathoI 1981;75:28-33. 10. Brittin GM, Dew SA, Fewell EK: Automated optical counting of blood platelets. Blood 1971;38:422-430.
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