Hematologic and molecular studies. (A) Peripheral blood smear (May-Grünwald-Giemsa). At low magnification (×100), anisocytosis and spherocytes are conspicuous (i). At high magnification (×1000), one sees spherocytes (ii), macrocytes (iii), hemighost (iv), and a nucleated red cell (v). (B) Restriction analysis of genomic DNA for G6PD-Med variant (Exon 6; 563 C>T; 188Ser>Phe).²⁵  After MboII digestion of an amplicon including exons 6, intron 6, and exon 7, the restriction fragments expected from wild-type (WT) G6PD (377 bp) and from mutant G6PD-Med allele (277 bp) are indicated. Controls (WT, heterozygote, and hemizygote) and patient samples are shown; the patient is heterozygous for G6PD-Med. MW, molecular-weight size marker 100 bp DNA ladder; the brighter band is 500 bp. (C) Complementary DNA restriction analysis of G6PD transcripts. Granulocytes (polymorphonuclear neutrophils [PMN]) were isolated by using the double-density centrifugation method; monocytes (Mø) were obtained by using an adherence technique from peripheral blood mononuclear cells. At the time of sampling, 99% of granulocytes and 97% of monocytes were PNH. Normal (non-PNH) lymphocytes (L) were obtained after monocyte-depletion of peripheral blood mononuclear cells. G6PD complementary DNA obtained from the selected blood cell populations was amplified, and an amplicon comprising exons 4 to 7 was digested with MboII. The restriction fragments relevant for the identification of normal (246 bp) and mutant G6PD-Med (147 bp) transcripts are indicated (the 208 bp fragment arises from either transcripts). The patient’s PNH granulocytes and monocytes expressed only the G6PD-Med transcript. The patient’s normal (non-PNH) lymphocytes also expressed predominantely the G6PD-Med transcript, suggesting that in this patient, X inactivation in hematopoietic cells had been highly skewed toward G6PD deficiency, regardless of PNH. MW, molecular-weight size marker 100 bp DNA ladder; the upper band is 500 bp.