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Ball Och 2008sdf
Ball Och 2008sdf
A . J . BALLOCH, A A I M L S & M . N . C A U C H I , M D ,
PhD, FRCPath
Department of Haematology, Royal Women’s Hospital, Grattan
Street, Carlton, Vicforia,Australia
PATIENTS
The results of full blood counts (FBC) were collected from 11 210 women tested
from the out-patient antenatal clinics of the hospital and 5519 non-pregnant
patients.
The full blood counts were matched with the patient’s hospital delivery
records. The gestation at delivery enabled calculation of the gestation at the date
Correspondence: A. Balloch, Haematology Department, Royal Women’s Hospital, Grattan Street,
Carlton, Victoria, 3053, Australia.
7
8 A . J . Balloch and M . N . Cauchi
of the full blood count. The gestation used in the calculation was that derived
from the date from the last menstrual period. If the gestation by ‘dates’ was not
available, the estimation by ultrasound or clinical criteria were used.
The data from antenatal patients were divided into three gestational groups.
First trimester defined as less than 14 weeks gestation. Second trimester defined as
14 to 26 weeks gestation and third trimester defined as greater than 26 weeks
gestation.
The first and second trimester data sets were seen to be biased toward the early
weeks due to the fact that the majority of women present early in their pregnancy
for antenatal testing. To rectify this bias, patients were randomly deleted to yield
equal numbers for each week within the first and second trimester data sets. The
third trimester data set was shown to be random (Runs test P < 0.01). This
yielded 3544 first trimester, 1794 second trimester and 2073 third trimester FBC
results.
Non-pregnant patient results were collected from gynaecology clinic and ward
locations. This yielded 5519 FBC results.
BLOOD S A M P L E S
Blood samples were collected into Sarstedt EDTA Monovette tubes (1.6 mg/ml
potassium EDTA).
FBC A N A L Y S I S
Samples were processed by a Coulter Counter S Plus I1 with 100 pl upgrade. The
counter was calibrated with fresh blood for all parameters except the MCV which
was calibrated against Coulter 4C normal control.
Ortho acuglobin haemoglobin standards were used with Drabkins solution for
haemoglobin calibration.
Reference cell counts were obtained from a Coulter ZBI single aperture
particle counter. Platelet rich plasma was used for platelet count calibration. The
ZBI counter was calibrated by the half count method, using the MCV of Coulter
4C normal control as the reference for threshold settings, as recommended by the
manufacturer.
Quality control was monitored with Coulter 4C normal control, Bulls (X,)
analysis of red cell indices and a local Haematology Instrumentation Control
Society (HICS) control material. External quality control consisted of HICS and
Royal College of Pathology of Australasia (RCPA) control material. Manual
leucocyte differential counts of 100 cells were performed.
DATA ANALYSIS
Data storage and handling was achieved with DBase Ill version 1.1 software.
Programs were written in DBase 111 programming language. Statistics were
calculated using Statgraphics version 2.6 software.
Haematology reference ranges in pregnancy 9
Table 1. Red cell parameters (mean 2 SD)
STATISTICAL METHODS
Bhattacharya plots were constructed for all parameters on the assumption that
haematology parameters can be described by either a single or mixture of
Gaussian distributions (Naus et al. 1980).
The observed frequencies were ‘smoothed’ before construction of the
Bhattacharya plots to minimize the effect of random fluctuations. The smoothing
procedure employed was that described by Savitzky & Golay 1964.
Linear regression analysis was performed on the linear portion of the
Bhattacharya plot. The mean and standard deviation were then calculated as
described by Naus et al. 1980. Where the Bhattacharya plot was found to be bent,
a Gamma curve was selected as a model for the parameters distribution. This was
in accordance with the selection criteria as recommended by Hemel et al. 1985.
The estimation of the R value (degrees of freedom) and 3, value (extension
factor) of the Gamma curves were performed as described by Hemel et al. 1985.
Where the Gamma model was chosen, the 2.5 and 97.5 percentile were
calculated from a chi square distribution (Hemel et al. 1985).
The 95% confidence intervals for the means of the red cell parameters were
calculated as described by Naus et al. 1980.
Results
The Bhattacharya plots revealed straight lines, consistent with Gaussian distribu-
tion, for all red cell parzmeters tested i.e., haemoglobin concentration (Hb),
haematocrit (Hct), red blood cell count (RBC) and mean cell volume (MCV). An
example of a Bhattacharya plot is shown in Figure 1 . The reference ranges, based
on the meank2 SD, are given in Table 1.
The Hb, Hct and RBC are seen to decrease throughout pregnancy consistent
with the expansion of plasma volume.
The 95% confidence intervals for the means of the red cell parameters are
shown in Table 2. The mean MCV shows a very small but statistically significant
rise from the first to the second trimester.
Bhattacharya plots of platelet count, white blood cell count, neutrophil count,
lymphocyte count and monocyte count were found to be bent for all patient
groups, see Figure 2.
10 A.J. Balloch and M.N. Cauchi
-2 I I I I I ,i I
70 75 80 a5 90 95 100 105
Mid point
Figure 1. Bhattacharya plot: first trimester MCV. The x axis represents the mid point of the class
interval. A 2 fl class interval was selected for the MCV. The mid point is plotted against log (f,+ ,/fJ
where f, is the frequency of class i. (Naus A. J. et al. 1980.) The arrows indicate the negative sloping
straight line which represent the portion of the data consistent with a Gaussian distribution.
Regression analysis was performed on this portion.
Table 2. 95% Confidence Intervals for the means of the primary red cell parameters
Mid point
Figure 2. Bhattacharya plot: first trimester neutrophil absolute number. The x axis represents the
mid point of the class interval. A 1.0 x 109/1class interval was selected for the neutrophil count. The
mid point is plotted against log (f,+ ,if,) where f, is the frequency of class i. (Naus A. J. et at. 1980.)
Thc absence of a negative sloping straight line suggests the distribution of neutrophil counts is not
well described by a Gamma distribution.
Log (1 + h/x)
Figure 3. Plot to check the Gamma model: first trimester WBC. Log (1 + h/x), where h is the class
interval and x is the mid point of the class, is plotted against F (A, R,X). The function F (A,R,X) is
calculated using estimates of R and 1 as described by Hemel et al. 1985. The plot reveals a straight
line, indicated by the arrows, suggesting the data is well described by a Gamma distribution.
12 A.J. Balloch and M . N . Cauchi
Discussion
Table 4. Eosinophil and basophil parameters (2.5 to 97.5 percentile range from raw data)
Table 5. Comparison of the first trimester reference ranges with ranges obtained
by conventional methodology
The fact that we were unable to describe our eosinophil and basophil data by
either Gaussian or Gamma distributions is possibly due to the relatively small
patient numbers we used.
Eosinophil and basophil counts have been reported to be well described by
Gamma distributions in a study using over 40000 patients. (Swaanenburg et ul.
1987).
All parameters, except lymphocyte count, show an increased variance and
consequently larger reference range in the third trimester. This is consistent with
reports of changes in the trends in parameters occurring in the third trimester.
The haemoglobin concentration is lowest at approximately 34 weeks and then
rises until term (Fleming 1975). Similarly there is a small rise in haematocrit
(Taylor & Lind 1976).
White cell count continues to increase into the third trimester until approxi-
mately 34 weeks after which there is a decline (Taylor & Lind 1976).
The total white cell count decline is paralled by a decline in the neutrophil
count until term (Bluck et al. 1990). Red cell counts decline until approximately
30 weeks and then increase. The MCV increases until approximately 30 weeks
then falls. In iron supplemented women, however, the MCV reportedly continues
to increase (Taylor & Lind 1976).
The broad third trimester ranges may result from these fluctuations and
variations. The derivation of reference ranges from our patient data provides
convenient, inexpensive and specific reference ranges for our antenatal
population.
Acknowledgements
References
BLUCKR., DIXON M., RAMAGE C. & BLACKLOCK H. (1990) A reference range for the haematological
changes of pregnancy. N . Z . J . Med. Lab. Technot. 44, 103 106
14 A . J . Balloch and M . N . Cauchi