From MIDWIFERY MATTERS, Issue No.106, Autumn 2005
BLOOD SAMPLES from antenatal patients at different stages of pregnancy are routinely
tested in the haematology and the blood transfusion departments. This article
describes the significance of the more routine test results which are reported
by most haematology laboratories and which are occasionally accompanied by comments
relating to an abnormal parameter or a significant finding when examining a
stained blood smear. Significant results from the blood transfusion department
normally involve the detection of blood group antibodies.
The main problem in dealing with results in the antenatal population is that
what is considered as abnormal in the general normal female population is regarded,
haematologically, as normal in pregnancy. However, in this article, reference
is made to the non-pregnant female in order to explain the significance and
the reason for some of the comments made on the result forms. The obstetric
unit, laboratory staff and the consultant haematologist would normally decide
on what tests to perform and what limits constitute abnormal values and these
are included in the laboratory Standard Operating Procedures (SOP). All laboratories
have their own SOPs for performing every test and these also include ranges
on the normal population which can vary depending on the equipment used but
are fairly standard for most haematology parameters.
Full Blood Count
The most common request we receive is for a full blood count (FBC). The important
parameters in this request are the haemoglobin (Hb or Hgb), the white blood
Cells (WBC) and platelets (Plts). Reports from most laboratories will have other
parameters such as RBC (red blood cells ), MCV (mean cell volume), MCH (mean
cell haemoglobin) and MCHC (mean cell haemoglobin concentration) on the result
form. These are known as 'Absolute Values' and reflect mathematically the size
and haemoglobin contents of a red cell (some analysers would also have other
red cell parameters available to staff on the computer screen). All these parameters
are mainly used by the laboratory staff when interpreting stained blood smears
and comments made on the report concerning red cells would therefore reflect
any significant abnormal value of these in the antenatal patient. The blood
smears are prepared and stained when results are outside an accepted range or
'flagged up' automatically by the analyser indicating that abnormal cells could
be present in the sample.
Most haematology laboratories have very sophisticated analysers that measure
all the parameters simultaneously. The haemoglobin is measured by 'bursting'
(haemolysing) the red cells and releasing the haemoglobin from the cells. This
is red and the density of the colour is measured and automatically converted
to a haemoglobin value.
Most analysers also measure the total red cell count and also the MCV, which
is the average volume of a red cell. This is important as a low MCV can indicate
iron deficiency and a raised result can indicate B12 and folate deficiency (again
the values vary in pregnancy). Absolute values such as the MCHC, mean cell concentration,
which is the percentage of an individual red cell volume occupied by haemoglobin
and MCH which the average haemoglobin content of the red cells are automatically
calculated within the analyser software. These values are therefore a guide
to iron deficiency. A typical iron deficiency blood picture would have a low
MCV and a reduced MCHC value together with other features such as a lack of
haemoglobin (also know as hypochromasia) in the red cells in the stained smear.
In order to complete the diagnosis, laboratories would request or suggest that
a sample be sent for a ferritin assay. Most of the body's iron is stored in
this form and this routine test gives an indication of the actual iron stores.
Other iron study tests are available but the haemoglobin value and appropriate
comments on the report form together with the further tests requested would
normally be adequate to make the diagnosis.
Haemoglobinopathy
Depending on their ethnic origin, antenatal patients may have a haemoglobinopathy
screen. This test detects the presence of variant/abnormal haemoglobin forms
or thalassaemia traits is investigated. However, these additional haematology
parameters (Absolute Values) can give an indication of whether there is a possibility
that thalassaemia is present and in some cases, depending on a pattern of red
cells on the analyser, whether abnormal haemoglobin forms are present. The normal
indication that an abnormality is present is when the MCV is lower than normal
(less than 80fl) and the mean cell haemoglobin (MCH) is less than 27pg. Some
laboratories will also have calculations built into their computer system to
highlight these abnormalities. Normally a smear would be prepared and examined
and a haemoglobinopathy and thalassaemia screen would be suggested together
with a ferritin check because of the low MCV.
Anaemia
In anaemic patients with a raised MCV the blood smear would be examined for
evidence of B12 or folic acid deficiency. In this case the haemoglobin would
be low, the MCV would be raised but the MCHC should be normal. Other features
would be noted in the stained smear, such as more lobes than normal in the white
blood cell neutrophil population. Larger than normal red blood cells would be
confirmed microscopically and also note would be made of a population of red
cells that can look like tear drops (poikylocytes) If these features were present,
B12 and folic acid assays test would be suggested to confirm the diagnosis.
Again the upper range for the MCV is increased in pregnancy but this would have
been considered before a comment is made. (Evidence of liver problem or hypothyroidism
would also have been considered when examining blood smear prepared from a non
pregnant anaemic patient with a raised MCV.)
White Blood Cells
The analysers also measure the total number of white blood cells present. Again
the range for women who are not pregnant is around 4.0 - 11.0 x109/L (that is
4-11,000 per cubic millimetre of blood) but this can increase to a much higher
level during pregnancy. Five different types of cells are normally identified
and measured. These are neutrophils which phagocytose bacteria, lymphocytes
that are involved with the immune mechanism, monocytes (a type of phagocytic
cell), eosinophils (the main reason for an increase would be an allergic reaction)
and basophils. In the normal population an increase in neutrophils (neutrophilia)
would suggest a bacterial infection and increased lymphocytes could suggest
a viral infection.
During the counting process haematology analysers can also flag abnormal cells.
These would then trigger the preparation and staining of a blood smear and this
is when in the routine situation that conditions such as glandular fever, leukaemia
and other haematological disorders are identified. Diagnosis is confirmed by
performing further confirmatory tests including referral to the consultant haematologist.
Platelets
Platelets are cells that play an important role in the coagulation process and
maintaining the integrity of the blood vessels. Thrombocytopaenia is the term
used to describe a lower than normal platelet count. A blood smear is always
prepared and examined for evidence of a clot, as this would reflect the quality
of the specimen as small clots in the sample can significantly reduce the platelet
count. Most laboratories would request a repeat sample together with a coagulation
screen. It is important to confirm the presence of low platelets as soon as
possible in pregnancy and continue to monitor the woman.
Grouping and Antibody Screen
Blood transfusion departments are routinely asked by antenatal clinics for blood
grouping and an antibody screen. Two blood groups systems are normally reported,
the ABO and the Rhesus. Most of the transfusion investigations involve antigen
and antibody reactions - a specific antigen will react with that specific antibody.
Antibodies are formed in response to a specific antigen, which are usually protein,
polysaccharide etc. in nature. Normally, the body produces these antibodies
through the immune mechanism (involving the lymphocytes) to any antigen that
is foreign to it (or non-self).
The blood group antigens are located on the surface of the red cells and any
antibodies produced is found in the plasma. Use is also made of this specific
antigen antibody reaction in the technology involved in transfusion. Known antibodies,
called antisera in the laboratory, are used to detect the unknown antigen on
the red cells. If a reaction occurs when a specific antisera is added to a patient's
red cells, then that antigen must be present on the surface of the cells and
therefore the blood group of that patient can be ascertained.
Reaction to Anti-A and Anti-B form the basis of the ABO grouping system (this
is a more complicated system, as it involves naturally occurring antibodies
in the plasma which is also tested for using known red cells (A and B antigens).
Rhesus positive patients have the D antigen on the surface of the red cells
and rhesus negative patients have not, so this group is determined by adding
known antibody D (Anti-D) to the patient's red cells which will detect the D
antigen in a Rhesus positive patient and no reaction will make the patient Rhesus
negative.
Antibody Screen test
The antibody screen test is a request to detect antibodies to any blood group
system. The ABO and Rhesus systems are the ones reported but there are numerous
others, examples are Kell, Kidd, Duffy. Basically, plasma should not contain
blood group antibodies (ABO is the only exception but these are not important
when compatible blood is transfused). If red cells containing antigens that
are foreign to the patient enter the circulation, this could be during transfusion
as only ABO and Rhesus is checked for compatibility or a trans-placental leak
during pregnancy, it is conceivable that the patient can produce antibodies
(although this depends on many factors). These antibodies will not cause any
problems during that episode but will circulate in the patient's circulation.
If a transfusion is required at a later date, only the ABO and rhesus group
will be performed, so if the red cells in the blood bag contain an antigen corresponding
to that antibody, a haemolytic reaction will take place in the patient's circulation.
Similarly, if the patient becomes pregnant, these antibodies can cross the placental
barrier and therefore react against the foetal red cells if they contain that
particular antigen.
The antibody screen test, screens the patient's plasma for all known clinically
important blood group antibodies. Commercially available red blood cells are
used which contain antigens all the important blood group systems and the patient's
plasma/serum is added to these cells. The method used in the laboratory involves
detecting a reaction between these cells and the patient's plasma/serum when
an antibody is present. The screen normally involves three batches of test cells
which between them contain all blood group antigens and the 'identification
panel' involves ten batches of cells and the staff will identify the antibody
depending on the pattern of the results.
In a situation where a transfusion is needed for a patient with an antibody,
known antibodies (antiserum) for that particular antigen would be used to screen
red cells from donor bags in order to find ones that showed no reaction to this
antiserum. This would confirm that the antigen was not present and no reaction
would take place in vivo if the red cells were transfused to the patient with
that particular antibody.
Direct Coombes
The direct Coombes test (DCT) on cord blood involves a similar principle. This
test involves looking for antibodies from the mother that are attached to the
baby's red cells i.e. the baby would have a blood group antigen on the red cells
surface that matches an antibody present in the mother's circulation. The method
used in the laboratory can identify the presence of these antibodies on the
surface of the baby's red cells.
Immunoglobulin Anti-D
The administration of immunoglobulin anti-D to rhesus negative mothers involves
an antibody antigen reaction. The mother is injected with antibody-D, no reaction
takes place with her own red blood cells as she is rhesus negative so there
is no D antigen present. However, if rhesus positive blood from the baby enters
her circulation, the antibody-D will react with the D antigen present on these
and they will be removed from the mother's circulation before her immune system
recognises them as foreign and therefore prevents her from producing an antibody
to the D antigen (anti-D).
Kleihauer test
The Kleihauer test performed on Rhesus negative mothers who give birth to Rhesus
positive babies involves the staining of the maternal blood for foetal cells.
The technique is based on the fact that the baby's haemoglobin molecule is different
from that of an adult (termed Hb F -foetal - as opposed to Hb A -adult). One
property of this haemoglobin is that it resists acid elution whereas adult does
not and this is utilised in the method. An eluting solution is added to a smear
of the maternal blood, the mother's red cells will be destroyed but the baby's
will resist this. A stain is added that stains all the red cells and any intact
cells must be foetal in origin. These are counted microscopically and reported.
There are other laboratory investigations that can be performed on antenatal
and postnatal women and also on cord samples but the tests described are more
of the routine tests requested in a normal uncomplicated pregnancy.
Dr G Rees is Lead Biomedical Scientist in Haematology and Blood Transfusion at the Carmarthenshire NHS Trust.
Updated LW January 17, 2007