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Hematology General 2 Kleihauer Test Test
Rationale The KB
test is the standard method of detecting fetal-maternal hemorrhage (FMH). It
takes advantage of the differential resistance of fetal hemoglobin to acid. A
standard blood smear is prepared from the mother's blood, and exposed to an
acid bath. This removes adult hemoglobin, but not fetal hemoglobin, from the
red blood cells. Subsequent staining makes fetal cells (containing fetal
hemoglobin) appear rose-pink in color, while adult red blood cells are only
seen as 'ghosts'. A large number of cells (over 5000) are counted under the
microscope and a ratio of fetal to maternal cells generated. The
Kleihauer test is a staining technique in which foetal cells can be
distinguished from adult red cells. It is an important test in the prevention
of Haemolytic Disease of the Newborn (HDN) due to anti-D allo-immunisation in
Rh(D) negative mothers. The test also has a diagnostic role in some
conditions such as stillbirth, extra cephalic version and abdominal trauma. All
babies of Rh(D) negative mothers are ABO and Rh grouped following delivery.
If the baby is found to be Rh(D) negative, no further tests are performed. If
the baby is found to be Rh(D) positive, it is important to determine if there
has been a Trans-Placental Haemorrhage (TPH) of baby's cells into the mothers
circulation at delivery. These cells must be removed before the mothers
immune system can recognise them as foreign and produce her own immune
anti-D. This immune anti-D, if allowed to develop, could cause HDN in
subsequent pregnancies. To prevent this an injection of anti-D gamma globulin
is given to the mother to "mop up" any foetal cells before her
immune system recognises them. Test Methodology To
prepare Kleihauer blood films, maternal blood is diluted in saline and spread
onto slides. These films are then fixed in 80% ethanol, eluted in acid
haematoxylin (the basis of this test being that foetal cells, as opposed to
adult cells, are resistant to acid denaturation) and counterstained in
erythrosine B. The films are examined microscopically to determine the size
of any TPH. The higher the foetal cell count, the larger the foetal bleed and
therefore the higher the dose of anti-D required. In those
with positive tests, follow up testing at a postpartum check should be done
to rule out the possibility of a false positive. This could be caused by a
process in the mother which causes persistent elevation of fetal hemoglobin,
e.g. sickle cell trait. The
Kleihauer test, along with the production of anti-D gamma globulin has
contributed significantly to a decrease in the number of babies suffering
from HDN due to immune anti-D.
Glycosylated (or
glycated) hemoglobin (hemoglobin A1c, Hb1c , or HbA1c, A1C) Form of hemoglobin used primarily
to identify the average plasma glucose concentration over prolonged periods
of time. It is formed in a non-enzymatic pathway by hemoglobin's normal
exposure to high plasma levels of glucose. Glycosylation of hemoglobin has
been implicated in nephropathy and retinopathy in diabetes mellitus.
Monitoring the HbA1c in type-1 diabetic patients may improve treatment. The
approximate mapping between HbA1c values and average blood glucose
measurements over the previous 4-12 weeks is shown in the table below.
Heparin induced thrombocytopenia
(HIT) Type
1 ~ 24hrs after heparin therapy, transient and mild Type
2: 5% of all patients >50%
reduction in platelets >5days
after starting Presents
with thrombosis IgG against
complex of heparin-platelet factor 4 (PF4) Diagnosis
by detecting antibody. Stop
heparin, Warfarin can cause skin necrosis, delay until anticoagulated Treat
with thrombin inhibitors (hirudin, lepirudin, argatroban) Post-transfusion purpura Severe thrombocytopenia
7-10 days after platelet infusion e.g. in RBC. Caused by anti HPA-1a (P1AI)
. Trasient may need immunoblobulins or plasma exchange. Blood donations: Collected into CPD. C= Citrate binds calcium, anticoagulates P=Phosphate D= Dextrose. Red cells stored at 4-6C stable foir 35 days Leuco-depletion occurs routinely. Reduces febrile
reactions, HLA alloimmunization, CMV transmission, new variant
Creutzfeld-Jacob disease. Each unit PRBC gives 200-250mg of iron. RBC can be strored in a frozen state. Platelet
transfusions: Stored at room temperature. Give to keep: Routine: pl>5 x 10E9/l Bleeding or surgery >50 Brain trauma, surgery eye surgery >100 Platelets express class l HLA antigens. Cryoprecipitate: Obtained by thawing FFP at 4C contains factor Vlll
and fibrinogen. Used for fibrinogen replacement in DIC. Neonatal
polycythemia Hct >0.65 Twin-twin, delayed clamping cord, intrauterine
growth retardation, maternal hypertension, and maternal diabetes. Fetomaternal
alloimmune thrombocytopenia Similar to hemolytic disease of the newborn (HDN). But occurs in the firs pregnancy in 50% unlike HDN. 1 in 1000-5000 Antibody against paternal inherited antigen (HPA-1a
in80%, Hpa-5b (PLATELET GLYCOPROTEIN Ia/IIa) in 15%)
Neonatal coagulation APTT and PT
prolonged due to reduced Vitamin K related coagulation factors (ll, Vll. lX,
X) normal at 6 months. Thrombosis die to
~60% level of protein C Rh HDN Fetal blood in
mother can be diagnosed by Kleihauer or PCR. Rh –ve mothers given
500U (100ug) of anti-D at 28 weeks and 34 weeks and birth for Rh+ve baby. Kleihauer test done
at birth +/- flow cytometry determines dose of anti-D (if >4ml) Erythroblastosis fetalis.
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