Objective: Immunophenotyping of blood lymphocytes is an important tool in the diagnosis of hematologic and immunologic disorders. Because of maturation and expansion of the immune system in the first years of life, the relative and the absolute size of lymphocyte subpopulations vary during childhood. Therefore we wished to obtain reference values for the relative and the absolute size of all relevant blood lymphocyte subpopulations in childhood. Study design:We used the lysed whole blood method for analysis of lymphocyte subpopulations in 429 blood samples from neonates (n = 20), healthy children (n = 358), and adults (n = 51). The following age groups were used: I week to 2 months (n = 13), 2 to 5 months (n = 46), 5 to 9 months (n = 105), 9 to 15 months (n = 70), 15 to 24 months (n = 33), 2 to 5 years (n = 33), 5 to 10 years (n = 35), and 10 to 16 years (n = 23).Results: Our results show that the absolute number of CD19* B lymphocytes increases twofold immediately after birth, remains stable until 2 years of age, and subsequently gradually decreases 6.5-fold from 2 years to adult age. The CD3* T lymphocytes increase 1.5-fold immediately after birth and decrease threefold from 2 years to adult age. The absolute size of the CD3*/CD4* T-lymphocyte subpopulation follows the same pattern as the total CD3 + population, but the CD3+/CD8 * T lymphocytes remain stable from birth up to 2 years of age, followed by a gradual threefold decrease toward adult levels. In contrast to B and T lymphocytes, the absolute number of natural killer cells decreases almost threefold in the first 2 months of life and remains stable thereafter. Our study also showed that changes in the absolute size of lymphocyte subpopulations are not always consistent with changes in their relative size. This demonstrates that the relative counts of lymphocyte subsets do not reflect their actual size and are therefore of limited value. Conclusion:On the basis of this study we strongly recommend that immunophenotyping of blood lymphocytes for the diagnosis of hematologic and immunologic disorders be based on the absolute rather than on the relative size of lymphocyte subpopulations. Our data can be used as age-matched reference values for blood lymphocyte immunophenotyping. (J Pediatr 1997; 130:388-93)

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Fifty‐five laboratories participated in a send‐out study of four peripheral blood samples comparing a standard protocol vs. local protocols for flow cytometric lymphocyte immunophenotyping. The standard protocol included centrally provided reagents, instrument setup using triple‐fluorescent microbeads and a three‐color, whole‐blood immunostaining technique based on fluorescein isothiocyanate and phycoerythrin‐labeled monoclonal antibodies, erythrocyte lysis, washing, fixation, and identification of nucleated cells by the DNA/RNA stain LDS‐751. Data analysis guidelines included lymphocyte selection using CD45,CD14‐assisted “backgating” on forward (FSC) and sideward (SSC) light scatter and placement of fluorescence (FL) markers on the basis of the isotype control staining. Most (i.e., 77%) of the variation in results of percentage lymphocyte subset assessments using the standard protocol was explained by laboratory, sample, background FL, and the interaction between laboratory and sample. Purity and completeness of the FSC,SSC lymphogate, background FL, flow cytometer type, and flow cytometer setup (which were either partly or entirely determined by laboratory) contributed significantly to the variation. The effect of the leukocyte differential count on the variation in absolute numbers of lymphocyte subsets was particularly large in lymphopenic samples. The use of this standard protocol vs. local protocols did not reduce the interlaboratory variation. Instrument incompatibility with the standard protocol (e.g., incompatible filter combinations for LDS‐751 detection) and lack of experience of many participants with three‐color flow cytometry (in particular with the use of LDS‐751) may have contributed to that result. We suggest that training and experience in a universally applicable standard protocol are critical for minimization of interlaboratory variation in flow cytometric immunophenotyping. Cytometry 30:166–177, 1997. © 1997 Wiley‐Liss, Inc.