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.

Monocytes were observed in the blood of inshore hagfish Eptatretus burgei. The monocytes were round or oval, high nucleus/cytoplasm ratio, and have several round or oval eosinophilic granules, which show dark red or blackish red color when stained with May-Grünwald (MG), Giemsa, and MG-Giemsa stain. The granules were positive for alkaline phosphatase, acid phosphatase, and α-naphthyl butyrate esterase. Also, the granules show metachromatic (reddish purple) with toluidine blue. However, ꞵ-glucuronidase, α-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase and peroxidase were not detected in the monocytes, and negative for periodic acid Schiff reaction, alcian blue, Sudan black B, Sudan III, and oil red O. The monocytes engulfed many yeast particles (zymosan).

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 granules (two-layer) were observed in the eosinophils (eosinophil granule. EG： type 1, EG1； type 2, EG2) of larva (ammocoetes) of far eastern brook lamprey Lethenteron reissneri collected in a tributary of the Koyagawa River in Yamaguchi Prefecture. The EG1 consisted of inner eosinophilic layer (L0) and chromophobic outer layer (L1). Dark (low light transmittance) inclusion structure (IS), which was various size and morphology (round, oval, rod, or spindle), was observed in the L0 of many EG1 (only one IS in a EG1). The IS was found in the cytochemical staining preparation, but not in the preparation stained with May-Grunwald (MG), Giemsa and MG•Giemsa. Therefore, recognition of IS was affected with the eosin-stained L0 of EG1. The EG1 classified into three subtypes (EG1a, EG1b and EG1c) based on the optical artificial image (OAI) of IS in L0. The EG1a had no OAI (probably no IS). The OAI of both EG1b and EG1c were larger than IS. The former was round or oval chromophobic area (OAI-1), and latter was expanded and rugged (threedimensional) image (OAI-2； chromophobic； round, oval, or rod). The EG1a may be prototype of EG1. The EG1 showed no positive reaction by various cytochemical stains. The EG2 had chromophobic inner layer (L0) and basophilic (orthomethylenophilic) outer layer (L1). Some enzymes (alkaline phosphatase, acid phosphatase, ꞵ-glucuronidase, α-naphtyl acetate esterase, naphthol AS-D chloroacetate esterase) were detected in L0 of EG2. The eosinophils lacked α-naphtyl butyrate esterase and peroxidase.

Two types of stratified [two-layer； inner layer (L0) and outer layer (L1)] granules were observed in the neutrophils (neutrophil granule, NG： type 1, NG1a； type 2, NG2) of larva (ammocoetes) of far eastern brook lamprey Lethenteron reissneri collected in a tributary of the Koyagawa River in Yamaguchi Prefecture. The NG1a consisted of chromophobic L0 and chromatophilic L1. On the other hand, the NG2 had chromatophilic L0 and chromophobic L1. The L1 of NG1a and L0 of NG2 showed a variety of colors [eosinophilic or basophilic (orthomethylenophilic or metaazurophilic)] depending on the staining conditions. These layers were positive for alkaline phosphatase, α-naphtyl acetate esterase and Sudan black B. Some enzymes, such as acid phosphatase, ꞵ-glucuronidase, and naphthol AS-D chloroacetate esterase were detected in the L0 of NG2. The neutrophils lacked α-naphtyl butyrate esterase and peroxidase.