Introduction

During normal pregnancy, the female body undergoes physiologic changes in almost every organ system to harbor the growing fetoplacental unit. The hematologic system is no exception to this convention as the maternal blood undergoes changes both in quantity as well as its constitution. The constituents of maternal blood are testable to screen and diagnose a wide variety of conditions relating to both healthy pregnancies as well as diseases associated with pregnancy.

A little before reaching full term, the volume of maternal blood is about 30% above baseline. The excess is likely due to increased levels of estrogen and aldosterone, which cause the kidneys to retain higher amounts of fluid. A higher number of erythrocytes accompanies this excess fluid with a slightly increased mean corpuscular volume in a healthy pregnancy.[1] Therefore, at delivery, there are between 1 to 2 liters of excess blood in the maternal circulatory system.

The prominent hematologic changes are physiologic anemia, expanded plasma volume, mild thrombocytopenia, a mild prothrombotic state, and in some individuals, mild neutrophilia. Other than the above changes, maternal blood also contains a wide number of other antigens, proteins, and hormones of clinical significance.

Issues of Concern

Certain findings fall outside the parameters of a normal physiologic pregnancy and require additional evaluation.

1. Non-physiologic anemia: A hemoglobin level of less than 10 mg/dl is concerning and requires evaluation. Iron deficiency is common in pregnancy due to the increased iron demand. Iron deficiency is evidenced by new microcytosis or iron studies demonstrating low iron stores.
2. Polycythaemia: Hemoglobin levels greater than 16 mg/dl should raise suspicion.
3. Leukocytosis: An increase in neutrophils can occur in some cases, even in the absence of inflammatory conditions or infection. Further hematologic evaluation is necessary in case of a white blood cell (WBC) count greater than 20000/microL, marked lymphocytosis, or a differential showing immature myeloid forms.
4. Leukopenia: An unexplained fall in the absolute neutrophil count below 1000/microL is concerning.
5. Thrombocytopenia: Gestational thrombocytopenia is a common occurrence during pregnancy with platelet counts in the range of 80000 to 149000/microL.[2] Severe thrombocytopenia or thrombocytopenia with bleeding require further evaluation.
6. Thrombocytosis: An increase in the platelet count of more than 500000/microL is unusual and requires investigation if unexplained.

Function

Maternal blood acts as the medium, which enables provision to the developing fetus of nutrients, gaseous exchange, and waste disposal. The placenta here serves as the gatekeeper of this exchange. Maternal blood floods the intervillous spaces, which is where the transfer takes place via both active and passive transport. Carbon dioxide, urea, uric acid, and creatinine are disposed into the maternal blood from the fetal circulation. Maternal blood contains IgG antibodies which diffuse through the placenta and provide naturally acquired passive immunity to the infant during the crucial first few months of life.

Related Testing

Initial Prenatal Screen

Prenatal care should be established in all women, ideally by ten weeks of pregnancy. Although the initial prenatal visit includes a history, physical examination, imaging studies, and blood work, in this section, we will only discuss the components of the prenatal evaluation that pertain to the maternal blood.

The tests commonly advocated in all patients are as follows:

• Hemoglobin, hematocrit, and mean corpuscular volume (MCV): To establish a baseline and screen for anemia.
• Rhesus type and ABO antibody screen: For the detection and prevention of alloimmunization and hemolytic disease of the newborn.
• Rubella and varicella immunity
• Syphilis, Hepatitis B, and HIV test
• Quadruple screen for fetal aneuploidies: This includes measuring maternal serum alpha-fetoprotein, beta-hCG, estriol, and inhibin A.

 For special at-risk patients, the following tests may be undertaken:

• Serum TSH, T3, T4: For thyroid dysfunction only if symptomatic, personal or family history of dysfunction, or an associated condition.
• Diabetes mellitus type 2: The American Diabetes Association (ADA) and the American College of Obstetricians and Gynecologists (ACOG) recommend testing in early pregnancy for undiagnosed type 2 diabetes in women with risk factors.[3][4]
• Toxoplasmosis serology: For at-risk patients.
• Hemoglobin electrophoresis: Undertaken for patients with a high-risk ethnic background or an MCV of less than 80 fL unrelated to iron deficiency
• Lead levels: For patients at risk based on history.

Clinical Significance

Serum Human Chorionic Gonadotropin

Human chorionic gonadotropin (hCG) is a hormone produced by the syncytiotrophoblast cells of the placenta after implantation. The primary function of hCG is to maintain the corpus luteum and thus progesterone secretion for the first 8 to 10 weeks of pregnancy, after which the placenta takes over the function of hormone production, and the corpus luteum degenerates. The hCG serves as an important biomarker for the detection of pregnancy, disease states associated with pregnancy, diagnosis of aneuploidies as well as a tumor marker for gestational trophoblastic disease, and germ cell tumors. The detection of the beta subunit hCG serves as the basis of the pregnancy test.

 Types of hCG Assay

• Immunometric sandwich-type assays are the routinely available commercial immunoassays for the quantification of serum hCG.
• Radioimmunoassays (RIAs): This is a competitive format assay based on the principle of competition between a fixed and known quantity of radiolabeled analyte and a sample analyte for a limited, fixed number of antibody binding sites.[5]

Conditions with Increased Levels of hCG

1. Multiple gestations
2. Hydatidiform moles
3. Choriocarcinomas
4. Down syndrome 

Conditions with decreased levels of hCG

1. Ectopic/failing pregnancy
2. Edwards syndrome
3. Patau syndrome

Anemia in Pregnancy

About 30 percent of women in the reproductive age group are anemic [6]. The World Health Organization (WHO) estimates that within the reproductive age group, the prevalence of anemia in pregnant women is even higher at around 40 percent. Given this high prevalence, recommendations for screening for anemia are at the first prenatal visit, with a repeat between 24 and 28 weeks of pregnancy.

As per the American College of Obstetricians and Gynecologists (ACOG) and the World Health Organization (WHO), anemia in pregnancy is defined as:[7][8][9]

• First trimester – Hemoglobin <11 g/dL
• Second trimester – Hemoglobin <10.5 g/dL
• Third trimester – Hemoglobin level <10.5 to 11 g/dL

Several causes lead to anemia in pregnancy, but chief among them are physiologic (dilutional) anemia of pregnancy and iron deficiency anemia.

• Physiologic anemia of pregnancy: This is the most common cause of anemia in pregnancy, though it is not a pathological condition. During normal pregnancy, there is a greater expansion of the plasma volume relative to the increase in the red cell mass, which results in dilutional anemia, termed as the physiologic anemia of pregnancy. Although this type of anemia is mild (hemoglobin of 10 to 11 g/dl), there is no specific hemoglobin or hematocrit value that can distinguish physiologic anemia from other causes of anemia in pregnancy.
• Iron deficiency anemia: It is the most common pathological cause of anemia during pregnancy. It may result from insufficient dietary intake, blood loss from menstruation or previous pregnancies, increased demand for iron during pregnancy, or impaired iron absorption from the gut.

Gestational Diabetes

Gestational diabetes has historically been defined as abnormal glucose tolerance with first onset or recognition during pregnancy.[10] Within the United States, the prevalence of gestational diabetes mellitus is roughly 6 percent.[11] Human placental lactogen secreted by the syncytiotrophoblasts of the placenta increases the overall insulin resistance. Since uncontrolled maternal blood glucose levels can result in several adverse outcomes for the mother and fetus, it is necessary to screen and treat gestational diabetes. 

The most common method is 2-step testing:

1. As a screening test, clinicians use the 50-gram one-hour glucose screen. The patient receives a 50-gram load of glucose irrespective of the last meal, followed in one hour by measuring the plasma glucose. As the threshold for a positive screen is increased from >130 mg/dl to > 140 mg/dl, there is a decrease in sensitivity and an increase in specificity.
2. The diagnostic test mainly used is the 100 grams three-hour oral glucose tolerance test. After an 8 hour overnight fast, an oral glucose load of 100 grams is administered, and serum glucose levels measured at fasting, and then every hour for 3 hours post glucose administration. The threshold values are as follows:

• Fasting: 90 mg/dl
• 1 hour: 180 mg/dl
• 2 hour: 155 mg/dl
• 3 hour: 140 mg/dl

A glucose concentration greater or equal to these values at two or more points is considered a positive test.

RhD Alloimmunization in Pregnancy

When Rh-D negative women deliver an Rh-D positive baby or have any mixing of Rh-D positive blood, the maternal immune system develops an antibody response to the Rh-D antigen. During any subsequent pregnancy, an Rh-D positive fetus or neonate is at risk of suffering from the hemolytic disease of the newborn due to the maternal immune system attacking the fetal red blood cells owing to the prior activation against this antigen. Due to the morbidity and mortality associated with this preventable condition, Rhesus typing and an antibody screen should be performed at the initial prenatal visit and then again at 28 weeks. The standard methods of screening maternal blood include:

• Indirect Coombs
• Gel micro-column assay
• Automated enzymatic methods

The Kleihauer–Betke is a special test performed on maternal blood used to calculate the required dosage of Rh-D immune globulin to inhibit the formation of Rh antibodies in the mother and prevent Rh disease in the future Rh-positive children. It measures the quantity of fetal hemoglobin transferred into the maternal blood and, based on that, gives an estimate of the dose of Rh-D immune globulin required.

Cell-free DNA (cfDNA) Testing

CfDNA is produced by the mother as well as the fetoplacental unit. There are two sources of fetal cfDNA in maternal plasma. It can be either from the apoptosis of the syncytiotrophoblasts (placental cells) or the apoptosis of fetal erythroblasts, which generate cfDNA in fetal circulation, after which the fragments cross the placenta into the maternal circulation. These fragments of fetoplacental cfDNA can be extracted from maternal blood and analyzed for abnormalities. Fetal cfDNA first appears in maternal blood at five weeks of gestation and is always present at around nine weeks of gestation.[12]

Indications: 

• Maternal age more > 35
• Abnormal maternal serum screening test
• Sonographic findings suggestive of fetal aneuploidy
• Fetal aneuploidy in a previous pregnancy
• Parental-balanced Robertsonian translocation

 Applications:

1. Trisomies: cfDNA has the highest sensitivity for detecting aneuploidies such as trisomy 21, 18, and 13. The detection rate (DR) for the aneuploidies is as follows:[13][14][15][16]

• Trisomy 21: 99.5 %
• Trisomy 18: 97.7 %
• Trisomy 13: 96.1 %

2. Sex chromosome aneuploidies: Although based on the same principle, the cfDNA can help to detect sex chromosome aneuploidies, the DR is much lower than for the common autosomal aneuploidies. 

3. Fetal sex determination 

Maternal Serum Alpha-fetoprotein (MSAFP)

Alpha-fetoprotein is a globulin synthesized in the fetal yolk sac, liver as well as gastrointestinal tract. Although its function is unclear, it may play a role in immunoregulation and act as an intravascular transport protein. AFP is secreted into the fetal urine by the kidneys and then excreted into the amniotic fluid. From here, it finds its way into maternal serum, and this maternal serum alpha-fetoprotein can be measured to screen and diagnose certain conditions. MSAFP is measured at 15 to 20 weeks of gestation (optimally at 16 to 18 weeks) to screen for fetal anomalies. 

 Increase MSAFP levels:

1. Open neural tube defects such as open spina bifida (meningomyelocele) or anencephaly 
2. Ventral wall defects like omphalocele and gastroschisis
3. Multiple gestations

 Decreased MSAFP levels:

1. Aneuploidies (Trisomy 21 and trisomy 18)

Liver Disorders of Pregnancy

There are a few disorders of the hepatobiliary system that are unique to pregnancy and are diagnosable with blood work.

1. Intrahepatic cholestasis of pregnancy: Presents with pruritis and jaundice in the late second or third trimester of pregnancy. Blood work shows elevated AST and ALT liver enzymes, bile acids, and bilirubin.
2. HELLP syndrome: This condition presents with nausea, vomiting, epigastric and abdominal pain, and hypertension in the late second or third trimester of pregnancy. Laboratory studies show elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and bilirubin, platelets counts below 100,000 and schistocytes on the peripheral smear.
3. Acute fatty liver of pregnancy: The patient has nausea, vomiting, anorexia, jaundice, and abdominal pain, usually in the third trimester of pregnancy. Some also have hypertension and proteinuria at presentation. Blood work demonstrates elevated AST, ALT, bilirubin, prothrombin time and international normalized ratio (PT/INR), and creatinine with decreased glucose and platelets.