1. Reference Intervals in Neonatal Hematology
Maria Proytcheva, MD University of Arizona
Banner University Medical Center-Tucson, USA

2. Financial Disclosure
The author has no conflict of interest to disclose

3. Do the Current Practices of Developing Neonatal Reference Intervals Meet Clinical Need?

4. Learning Objectives
Review the human hematopoietic ontogeny and normal hematologic values in newborns and neonates
Examine reference intervals in neonatal hematology and how they currently affect clinical practice
Provide a new approach for applying pediatric reference intervals in laboratory hematology

5. Hematopoietic System Ontogeny
There are age-specific quantitative changes that parallel age-specific functional differences in cellular function from birth to adulthood. Hypothetical model of human hematopoietic ontogeny based on amphibian, avian, murine, and human developmental data. The yolk sac provides two transient populations of committed progenitors that are thought to arise from a mesoderm-derived hemangioblast (HB) precursor. The first wave of progenitors produces primitive erythroblasts (PRIM. ERY.) (see text). The second wave produces burst-forming unit–erythroid (BFU-E) and several myeloid progenitors (MP) that seed the liver. Long-term hematopoietic stem cells (HSCs) arise later in the aorta-gonad-mesonephros (AGM) region that subsequently populate the liver and ultimately the marrow to generate the full panoply of definitive hematopoiesis. The HSCs from liver also provide naïve lymphoid cells to the thymus, and T-lineage maturation occurs there.
Kaushansky K et al.. In Williams Hematology, 9e; 2015;
Copyright © 2017 McGraw-Hill Education. All rights reserved

6. Fetal Blood
The composition depends on gestational age. The most dramatic changes occur between 2nd and 3rd trimester
Hgb, Hct, WBC, platelets increase while MCV and MCH decrease with advance of gestational age
Rate of increase from 22 to 40 weeks (Jopling, J., E. et all, 2009:Pediatrics 123: e )
Hgb ↑ 0.21 g/dL and Hct ↑ 0.64% per week
MCV ↓ from 119 fL at <25 weeks to 106 fL at 40 weeks
At 10 weeks, Hgb is 9.0 g/dL ( )
At term, cord blood Hgb 16.8 g/dL ( )
There are age-specific quantitative changes that parallel age-specific functional differences in cellular function from birth to adulthood

7. Fetal White Blood Cells and Platelets
2nd trimester: 2.0x109/L
Mid-3rd trimester: 4.0 x109/L
80-85% lymphocytes
5-10% neutrophils
Platelets peak at 150x109/L at 15 weeks of gestation and remain stable
Henry, E. and R. D. Christensen (2015). "Reference Intervals in Neonatal Hematology." Clin Perinatol 42(3):

8. At Birth WBC is high with relative neutrophilia
Mean cord blood Hgb at term 16.8 g/dL and MCV is high, 119 fL ( )
Factors affecting Hgb at birth and thereafter:
Degree of maturity
Time of umbilical cord clamping - early vs. late
Hemoconcentration
Sample source: capillary blood has higher
HCT than venous blood
WBC is high with relative neutrophilia
Platelet count is compatible to adults
Hemoconcentration due to movement of plasma from intra- to extravascular space in the first several hours of life

9. Hgb Dynamics in the First 28 Days of Life
17.5 g/dL ( )
13.0 g/dL ( )
The Hgb concentration decreases after birth, more significantly during the first 28 days of life
Significant drop in the first 8 day followed by gradual decrease to the end of the neonatal period
Erythropoiesis:
From low O2 saturation with high demand to high oxygen saturation with lower demand
Decrease RBC survival during the neonatal period
Henry, E. and R. D. Christensen (2015). "Reference Intervals in Neonatal Hematology." Clin Perinatol 42(3):

10. Premature Neonates Have Lower Hemoglobin
FT:
17.5 g/dL ( )
16.8 g/dL ( )
FT:
13 g/dL ( )
11.6 g/dL (8.2 to 15.8)
(29 to 34 Wks.)
Lower concentration at birth and more dramatic decline at the end of the neonatal period -
Henry, E. and R. D. Christensen (2015). "Reference Intervals in Neonatal Hematology." Clin Perinatol 42(3):

11. ANC in the First 3 Days of Life (Term)
Numerous studies demonstrate characteristic dynamics in ANC after birth
Peak occurs at 8 to 12 hrs. then gradually drops by the end of the 3rd day
Manroe, B. L. et al (1976). Journal of Pediatrics 95(1): 89-98
Mouzinho, A. et al (1994) Pediatrics 94(1):
Henry, E. and R. D. Christensen (2015) Clin Perinatol 42(3):
The mean neutrophil count at the end of day 3 is below the low range of ANC for 8 to 12 hours of age and the mean at the peak is above the upper level at day 3.

12. ANC in the First 3 Days of Life: Full Term vs. Premature Neonates
>36 weeks
28-to-36 weeks
Neutropenia – ANC <2,500/uL in term and <1,000/uL in preterm neonates at 72 hours
Schmutz, N. et al (2008). "Expected ranges for blood neutrophil concentrations of neonates: the Manroe and Mouzinho charts revisited." J Perinatol 28(4):

13. Summary of Neonatal Hematology
Age-dependent dynamics affect blood composition during the first 28 days of life
The changes are gradual and values are significantly different at the beginning and end of the neonatal period
Premature newborns and neonates have lower Hgb, HCT, ANC and higher MCV as compared to term neonates
Reference intervals are needed to distinguish normal from abnormal to support clinical decision-making and assure adequate management of individual patients

14. Reference Intervals (RIs)
RIs are obtained by measuring the values in a reference group (assumed healthy) and taking two standard deviations on either side of the mean
5th and 95th percentiles of a reference population’s distribution (spanning 90% of the total population)
RIs are not the same as normal ranges (measurements in known healthy volunteers)
2.5th and 97.5th percentiles of a healthy population’s distribution (spanning 95% of the total population)
When assuming a normal distribution, the reference range is obtained by measuring the values in a reference group and taking two standard deviations either side of the mean. This encompasses ~95% of the total population.
The area under this curve within ±1 σ from the mean is approximately 68.2% of the total area, meaning that 68.2% of data points from a Gaussian distribution should fall within ±1 σ of the mean. Similarly, 95.5% of the data points will be within ±2 σ of the mean, and 99.7% will be within ±3 σ of the mean.

15. CLSI EP28-A3C Guideline Applicable to Adults (age 18-to-65 years)
Establish a reference interval
Analyze at least 120 samples, age and sex specific
Verify RIs established elsewhere
Transfer RIs from another study
Verify with as few as 20 samples from qualified reference individuals
How to apply CLSI EP28-A3C Guideline to develop hematology RIs for neonates?
They don’t apply to neonates
The static and apply
The hematologic changes are continuous so the reference intervals need to reflect this spectrum of changes in comparison with the currently used reference intervals may result in substantial reduction in the number of samples considered pathologic
* Horowitz, GL, Chairholder, et al. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory;
Approved Guideline, Third Edition, EP28A3C (10/01/2010)

16. Published Studies (1976-2016) Monroe, B. et al (1976)
Dallas, USA, 585 samples from 304 normal and 320 samples from 130 neonates with complications
Mouzinho, A. et al (1994)
Dallas, USA ; 1788 samples from 193 small for gestational age newborns <1,500 gm
Brugnara, C. et al (2007)
Boston, USA; compared 3 hematology analyzers (Coulter STKS, Advia 120, Sysmex XE-2100)
Christensen, R. et al, ( )
Salt Lake City, USA, 350,000 samples from >100,000 children
Zierk, J. et al (2015)
Bremen, Germany; 167,000 samples from 32,000 children
Grecu et al (2013)
Timisoara, Romania; 845 samples
Conclusions:

17. Agreed Upon Conclusions About RIs for Neonates
With transition from fetal to adult hematopoiesis, there are dynamic, predictable, and reproducible changes over time in Hgb, Hct, MCV, RDW, absolute neutrophil and platelet counts
The changes are gradual and contiguous
Zierk, J. et al (2015): Clin Chem, 61:7;

18. Differences Between Studies
Different groups report different means and range values. The result is that there are no standard means and ranges available to laboratories at present
Schmutz
Schmutz
Schmutz
Monroe’s study includes babies with Down and other trisomies, known to have higher ANC or low ANC such as pregnancy induced hypertension, early onset of bacterial sepsis, or congenital neutropenia) Schmutz excludes those. Different hematology analyzers – Beckamn Coulter LH750 vs. Sysmex. Dallas (500 feet ASL vs. SLC 4800 ASL)
Schmutz, N. et al (2008); J Perinatol. 28(4): ; Manroe, B. L. et al (1976): Journal of Pediatrics 95(1): 89-98; Mouzinho, A. et al (1994). Pediatrics 94(1):

19. Present Practice of Developing RIs for Neonates
Identify published RIs with population closest to the population your laboratory serves
Define time-points for a specific analyte, i.e. Hgb, MCV, WBC, ANC, ALC, etc.
Validate the RIs using at least 20 individuals for each time-point

20. Problems with Current Laboratory Practices
There is no agreement of age-specific time points, which leads to significant variations in laboratory practices
Having few age-specific time points for neonates fails to reflect the continuous changes that occur during the transition from fetal to postnatal life
As a result, there is high risk of classifying healthy neonates as sick and vice-versa
Zierk, J. et al found misdiagnosis in 38% of hemoglobin samples
Zierk, J et al (2015).; Clinical Chemistry 61(7):

21. Comparison of Hgb RIs Used by 2 Laboratories
Age
Lab A
(Hgb g/dL)
Lab B
0-2 wks.
<1 day
1-6 days
7-13 days
2 wk.-1 mo.
Henry, E. and R. D. Christensen (2015). "Reference Intervals in
Neonatal Hematology." Clin Perinatol 42(3):

22. Defined Time-Point Approach
Day 1: 17.8 ( )
Day 2: 17.1 ( )
Day 3: 16.8 ( )
Day 4: 16.2 ( )
Day 5: 16.0 ( )
Day 6: 15.8 ( )
Day 7: 15.3 ( )
Day 14: 14.4 ( )
Day 21: 14.0 ( )
Day 28: 13.0 ( )
RIs Lab A
Ris Lab B
Henry, E. and R. D. Christensen (2015). "Reference Intervals in Neonatal Hematology." Clin Perinatol 42(3):

23. Comparison of ANC RIs Used by 2 Laboratories
Age
Lab A
ANC 103/µL
Lab B
<24 hrs.
0-3 d.
1-6 d.
3 d. - 2 wks.
1 wks. – 2 wks.
2 wks. -1 mo.
ANC of full term neonates during the 1st 72 hrs.**
Henry, E. and R. D. Christensen (2015). "Reference Intervals in Neonatal Hematology." Clin Perinatol 42(3):

24. Need for New Paradigm to Establish RIs in Neonatal Hematology
Develop Large Database(s) for RI in Neonatal Hematology by adding to the already published studies
Make actual data of the published old and new RIs publicly available
Include information on gestational age, ethnic composition of the population in the database, altitude of the area, type of samples used, type of hematology analyzers, and other pertinent data
Need for major grant funding to develop such databases
Individual lab will verify RIs from the appropriate database using as few as 20 samples from qualified individuals

25. Need to Develop a New Strategy for Result Displaying and Reporting
Develop a new approach for implementing RIs in neonatal hematology, i.e. using nomograms defining lower and upper limits of a hematology analyte, in the Laboratory Information System
There may be other effective approaches, too

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