Supplemental Question Discussion for 2005 LM-A.
T hi m aii i uded the folow i suppl ental questi regardi the O D 4 5 0 test. s lng ncl l ng em on ng W hen perform i anal s for am ni c fl d bii n, how does your l ng ysi oti ui lrubi aboratory correct for potenti bl al ood i nterference? A . C hl oroform extracti on B . M athem ati (e.g., A len) correcti cal l on/graphi ng C . O ther correcti on; pl ease speci fy D . W e do not perform a correcti on T he di buti of responses i show n i the folow i graph. stri on s n l ng
Correction Methods Used
50 4 5 Num ber ofLabs Using Method 4 0 3 5 3 0 2 3 2 5 2 0 2 0 1 5 1 0 5 0 CHL OROF ORM E RACTON XT I MAT CORRE I H CTON/ GRAP NG HI OT R CORRE I HE CTON DO NOT P RF E ORM CORRE I CTON 6 Correction Methods Used 4 4
Correction Method
T hese resul i cate that there i no cl consensus on the m ethod to correct for bl ts ndi s ear ood i nterference, or even that a correcti i necessary. S i on s nce cri cal pati ti ent m anagem ent deci ons m ay be based on the si O D 4 5 0 test, l aboratori m ust have a consi es stent, clni l rel i caly evant procedure to deal w i thi probl . th s em ntrauteri hem ol s i ne ysi n Liey descri l bed the basi for the O D 4 5 0 test.1 O ne can assess the degree of i s hem ol c di yti sease of the new born (H D N ) by m easuri the absorbance of bii n pi ents i am ni c ng lrubi gm n oti fl d ( O D 4 5 0 ) and cl ui assi ng the resul based on gestati fyi ts onal age i nto l ow ri i sk, nterm edi ate ri and sk, hi ri zones. Q ueenan proposed an al gh sk ternati ri categori on schem e based on the O D 4 5 0 test.2 ve sk zati W i both system s, the severi of H D N i m ore accuratel predi th ty s y cted w i seri O D 4 5 0 m easurem ents. th al 3 A typi procedure for O D 4 5 0 i descri cal s bed by A shw ood. B ecause am ni ocentesi i an i s s nvasi ve procedure, there i a ri that the am ni c fl d speci en m ay be contam i s sk oti ui m nated w i hem ogl n, w hi th obi ch 2
has an absorbance peak at 410 nm. The amniotic fluid specimen should be centrifuged before spectrophotometric scanning to remove red blood cells or other particulate matter. I there is an f oxyhemoglobin peak at 410 nm, one can correct the OD450 by subtracting 5% of the OD410. An alternative correction for the presence of hemoglobin in the amniocentesis specimen is extraction of the bilirubin fraction into chloroform before performing the scanning spectrophotometry.4,5 However, chloroform extraction reduces the OD450, even when no hemoglobin is present.6 I chloroform extraction f is used, all specimens should be subj ected to chloroform extraction, not j ust the specimens that are visibly contaminated with blood. Otherwise, OD450 results obtained on two serial amniotic fluid specimens from the same patient, one of which was extracted and the other of which was not extracted, may lead to an incorrect interpretation about the trend of the OD450 measurements and the clinical course of the HDN. Furthermore, since the Liley graph that is used to interpret the OD450 result was created using unextracted material, some authors have suggested that a new Liley graph should be constructed using chloroform-extracted material.6
David S. Wilkinson, MD, PhD Chemistry R esource Committee
References: 1. Liley AW. Liquor amnii analysis in the management of the pregnancy complicated by rhesus sensitization. Am J Obstet Gynecol. 19 61;8 2:1359 -7 0. 2. Queenan JT, Tomai TP, U ral SH, K ing JC. Deviation in amniotic fluid optical density at a wavelength of 450 nm in R h-immunized pregnancies from 14 to 40 weeks’ gestation: a proposal for clinical management. Am J Obstet Gynecol. 19 9 3;168 :137 0-6. 3. Ashwood ER . Determnation of A450 in amniotic fluid. I Burtis CA, Ashwood ER , eds. Tietz n: rd textbook of clinical chemistry, 3 ed. Philadelphia: WB Saunders, 19 9 9 :17 68 -7 0. 4. Hochberg CJ, Witheiler AP, Cook H. Accurate amniotic fluid bilirubin analysis from the "bloody tap". A preliminary report. Am J Obstet Gynecol. 19 7 6;126:531-4. 5. Spinnato JA, R alston K K , G reenwell ER , Marcell CA, Spinnato JA 3rd. Amniotic fluid bilirubin and fetal hemolytic disease. Am J Obstet Gynecol. 19 9 1;165:1030-5. 6. Foster K , Moore J, Hankins K , Parvin CA and G ronowski. Effect of blood contamination on delta 450 bilirubin measurement: an in vitro comparison of two corrective methods. C li C hem. 2004;50:1420-2. n
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