Neutrophil granules (NG) of West African lungfish Protopterus annectens were classified into two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules (type A, NG-A; type B, NG-B). The L1 of NG-A and both layers of NG-B were chromophobic, and L0 of NG-A showed polychromatophilic [eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)]. The L0 of NG-A showed metachromatic (reddish purple) with toluidine blue. On the other hand, L0 of NG-B were positive for acid phosphatase, α-naphthyl acetate esterase, α-naphthyl butyrate esterase and naphthol AS-D chloroacetate esterase. Both types of granules were negative in periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. Alkaline phosphatase, β-glucuronidase, and peroxidase were not detected in either granule.

Based on the findings of past literature, we speculated the existence of two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules in the neutrophils (granulocytes; blood cells) of amphioxus (Cephalocordata), especially Branchiostoma japonicum. Type 1 neutrophil granules (NG1a) consist of chromophobic L0 and chromatophilic L1. Other type (type 2; NG2) have chromatophilic L0 and chromophobic L1. The L1 of NG1a and L0 of NG2 show metaazurophilic (purple to blackish purple) in Giemsa staining preparation. Ultrastructurally, these two granule types will not be separately identified. These granules contain intragranular particles (granulons, g) in the L0 of granules. At least, three types of granulons (g-1, g-2, and g-3) are recognized in a granule and localized in L0. Two of them form an agglomerate with short rod shape and two layers (inner layer consisted by g-2, and outer layer by g-3). The agglomerate have been considered as tubular structure or microtubule (misinterpretation). Neutrophils of cyclostome (hagfish and lamprey) also have NG1 [NG1a or NG1b (chromophobic L0 and L1)] and NG2. Further, neutrophil granules of cyclostome also contain granulons (without forming of agglomerate).

Neutrophil granules (NG) of adult (mature) far eastern brook lamprey Lethenteron reissneri [L. sp. S (souther form)] were classified into two types of stratified [two-layer; inner layer (L0) and outer layer (L1)] granules (type 1, NG1a; type 2, NG2), like as larva (ammocoetes) of this species. The L0 of NG1a and L0 of NG2 in adult neutrophils were chromophobic, and L1 of NG1a and L0 of NG2 showed chromatophilic. The stainability of those chromatophilic layers with May-Grünwald (MG), Giemsa, or MG-Giemsa were varied [chromophobic, eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)] with a slight difference to larva. As seen in larval neutrophils, these layers were positive for alkaline phosphatase,α-naphtyl acetate esterase and Sudan black B, and some enzymes (acid phosphatase, ꞵ-glucuronidase, and naphthol AS-D chloroacetate esterase) were detected in the L0 of NG2. Alpha-naphtyl acetate esterase was positive in L0 of NG2 of adult neutrophils. Both larval and adult neutrophils lacked peroxidase.

Two types of stratified (two-layered) granules (type 1, NG1a; type 2, NG2) in the neutrophils of inshore hagfish Eptatretus burgei showed eosin-positive (positive site: outer layer (L1) of NG1a and inner layer (L0) of NG2). In contrast, both eosin-positive sites have been reported to be basophilic. From present result, it is clarified that both sites exhibit as follows: L1 of NG1a, eosinophilic and mataazurophilic; L0 of NG2, eosinophilic, orthomethylenophilic and mataazurophilic. The inshore hagfish neutrophils phagocytosed zymosan particles, in vitro. All blood cells showing phagocytosis were identified as neutrophil.