—  SHORT COURSE  —

Attilio Orazi, M.D., FRCPath. and Dennis P. O'Malley, M.D.

HEMOGLOBIN Anti-human hemoglobin is a polyvalent rabbit antibody reactive with normal adult erythrocytes. The antibody reacts only with human red cells and does not react with hemoglobin from other species. The antibody reacts with hemoglobin A, with hemoglobin A2 and also with hemoglobin F. By immunohistochemistry, the reagent stains red cell precursors. Normoblasts stain intensely. However, the sensitivity is great enough that pronormoblasts and erythroblasts stain also. Mature red blood cells do not stain strongly, probably due to the poor penetration of the antibodies. There is no other staining of any other cells. It is more effective if a heat-induced epitope retrieval technique is used. The stain works well both in formalin fixed and B5 fixation and is not materially inhibited by decalcification. I have found the anti-hemoglobin reagent to be the one of choice in staining of red cell precursors, and prefer it to glycophorin A. Representative cases in which the hemoglobin immunostaining is particularly useful include: The detection of small numbers of scattered erythroblasts and their differentiation from lymphocytes (e.g. aplastic anemia). Differentiation of normoblasts from lymphocytes in such cases as pediatric bone marrows, in which lymphocytes are numerous. The distinction between clusters of repopulating red cell precursors and residual clusters of leukemic myeloblasts in marrows with leukemia at the end of induction or beginning of consolidation. The distinction between early red cell precursors and poorly differentiated metastatic carcinoma. The recognition of megaloblasts in patients with megaloblastic anemias. The quantitation of red cell precursors in pediatric patients thought to have pure red cell aplasia. References Crocker J, Gyde OHB, Jenkins R: Demonstration of normoblasts in tissue sections by means of an immunohistochemical technique for haemoglobin. J Clin Pathol 1984;37:1312. Greaves MF, Sieff C, Edwards PA: Monoclonal antiglycophorin as a probe for erythroleukemias. Blood 1983;61-645. Neiman RS: Erythrblastic transformation in myeloproliferative disorders. Confirmation by an immunohistologic technique. Cancer 1980;46:1636. Pinkus GS, Said JW: Intracellular hemoglobin - a specific marker for erythroid cells in paraffin sections. An immunoperoxidase study of normal, megaloblastic, and dysplastic erythropoiesis, including erythroleukemia and other myeloproliferative disorders. Am J Pathol 1971;102:308.